IGF antagonist peptides

ABSTRACT

Peptides are provided that antagonize the interaction of IGF-1 with its binding proteins, insulin receptor, and IGF receptor. These IGF antagonist peptides are useful in treating disorders involving IGF-1 as a causative agent, such as, for example, various cancers.

RELATED APPLICATIONS

This application is a divisional application of co-pending applicationSer. No. 10/098,093 filed on Mar. 13, 2002, which is a non-provisionalappication filed under 37 CFR 1.53(b)(1), claiming priority under 35 USC119(e) to provisional application No. 60/275,904 filed Mar. 14, 2001,the contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to peptides that antagonize insulin-likegrowth factor (IGF), in particular, IGF-1. These peptides are useful intreating disorders caused or mediated by IGFs, such as cancer.

2. Description of Related Disclosures

There is a large body of literature on the actions and activities ofIGFs (IGF-1, IGF-2, and IGF variants). Human IGF-1 is a 7649-daltonpolypeptide with a pI of 8.4 (Rinderknecht and Humbel, Proc. Natl. Acad.Sci. USA, 73: 2365 (1976); Rinderknecht and Humbel, J. Biol. Chem., 253:2769 (1978)) belonging to a family of somatomedins with insulin-like andmitogenic biological activities that modulate the action of growthhormone (GH) (Van Wyk et al., Recent Pros. Horm. Res., 30: 259 (1974);Binoux, Ann. Endocrinol., 41: 157 (1980); Clemmons and Van Wyk, HandbookExp. Pharmacol., 57: 161 (1981); Baxter, Adv. Clin. Chem. 25: 49 (1986);U.S. Pat. No. 4,988,675; WO 91/03253; WO 93/23071). IGFs arestructurally similar to insulin, and have been implicated as atherapeutic tool in a variety of diseases and injuries.

The IGF system is also composed of membrane-bound receptors for IGF-1,IGF-2, and insulin. The Type 1 IGF receptor (IGF-R) is closely relatedto the insulin receptor in structure and shares some of its signalingpathways (Jones and Clemmons, Endocr. Rev., 16: 3-34 (1995)). The IGF-2receptor is a clearance receptor that appears not to transmit anintracellular signal (Jones and Clemmons, supra). Since IGF-1 and IGF-2bind to IGF-1R with a much higher affinity than to the insulin receptor,it is most likely that most of the effects of IGF-1 and IGF-2 aremediated by IGF-1R (Humbel, Eur. J. Biochem. 190:445-462 (1990); Ballardet al., “Does IGF-I ever act through the insulin receptor?”, in Baxteret al. (Eds.), The Insulin-Like Growth Factors and Their RegulatoryProteins, (Amsterdam: Elsevier, 1994), pp. 131-138). The crystalstructure of the first three domains of IGF-1R has been determined(Garrett et al., Nature, 394, 395-399 (1998)).

IGF-R is a key factor in normal cell growth and development (Daughadayand Rotwein, Endocrine Rev., 10:68-91 (1989)). Increasing evidencesuggests, however, that IGF-R signaling also plays a critical role ingrowth of tumor cells, cell transformation, and tumorigenesis (Baserga,Cancer Res., 55:249-252 (1995); for a review, see Khandwala et al.,Endocr. Rev., 21: 215-244 (2000)). Key examples include loss ofmetastatic phenotype of murine carcinoma cells by treatment withantisense RNA to the IGF-1R (Long et al., Cancer Res., 55:1006-1009(1995)) and the in vitro inhibition of human melanoma cell motility(Stracke et al., J. Biol. Chem., 264:21554-21559 (1989)) and of humanbreast cancer cell growth by the addition of IGF-1R antibodies (Rohliket al., Biochem. Biophys. Res. Commun., 149:276-281 (1987)).

The IGFs are potent breast cancer cell mitogens based on the observationthat IGF-1 enhanced breast cancer cell proliferation in vitro (Cullen etal., Cancer Res., 50:48-53 (1990)). Breast cancers express IGF-2 andIGF-R, providing all the required effectors for an autocrine-loop-basedproliferation paradigm (Quinn et al., J. Biol. Chem., 271:11477-11483(1996); Steller et al., Cancer Res. 56:1761-1765 (1996)). Because breastcancer is a common malignancy affecting approximately one in every eightwomen and is a leading cause of death from cancer in North Americanwomen (LeRoith et al., Ann. Int. Med 122:54-59 (1995)), new rationaltherapies are required for intervention. IGF-1 can suppress apoptosis,and therefore cells lacking IGF-1Rs or having compromised IGF-1Rsignaling pathways may give rise to tumor cells that selectively die viaapoptosis (Long et al., Cancer Res., 55:1006-1009 (1995)). Furthermore,it has recently become evident that alterations in IGF signaling in thecontext of other disease states, such as diabetes, may be responsiblefor exacerbating the complications of retinopathy (Smith et al.,Science, 276:1706-1709 (1997)) and nephropathy (Homey et al., Am. J.Physiol. 274: F1045-F1053 (1998)).

The IGF binding proteins (IGFBPs) are a family of at least six proteins(Jones and Clemmons, supra; Bach and Rechler, Diabetes Reviews, 3: 38-61(1995)), that modulate access of the IGFs to the IGF-1R. They alsoregulate the concentrations of IGF-1 and IGF-2 in the circulation and atthe level of the tissue IGF-1R (Clemmons et al., Anal. NY Acad. Sci.USA, 692:10-21 (1993)). The IGFBPs bind IGF-1 and IGF-2 with varyingaffinities and specificities (Jones and Clemmons, supra; Bach andRechler, supra). For example, IGFBP-3 binds IGF-1 and IGF-2 with asimilar affinity, whereas IGFBP-2 and IGFBP-6 bind IGF-2 with a muchhigher affinity than they bind IGF-1 (Bach and Rechler, supra; Oh etal., Endocrinology, 132, 1337-1344 (1993)).

In most cases, addition of exogenous IGFBP blunts the effects of IGF-1.For example, the growth-stimulating effect of estradiol on the MCF-7human breast cancer cells is associated with decreased IGFBP-3 mRNA andprotein accumulation, while the anti-estrogen ICI 182780 causes growthinhibition and increased IGFBP-3 mRNA and protein levels (Huynh et al.,J. Biol. Chem., 271:1016-1021 (1996); Oh et al., Prog. Growth FactorRes., 6:503-512 (1995)). It has also been reported that the in vitroinhibition of breast cancer cell proliferation by retinoic acid mayinvolve altered IGFBP secretion by tumor cells or decreased circulatingIGF-1 levels in vivo (LeRoith et al., Ann. Int. Med., 122:54-59 (1995);Oh et al., (1995), supra). Contrary to this finding, treatment of MCF-7cells with the anti-estrogen tamoxifen decreases IGF-R signaling in amanner that is unrelated to decreased IGFBP production (Lee et al., JEndocrinol., 152:39 (1997)). Additional support for the generalanti-proliferative effects of the IGFBPs is the striking finding thatIGFBP-3 is a target gene of the tumor suppressor, p53 (Buckbinder etal., Nature, 377:646-649 (1995)). This suggests that the suppressoractivity of p53 is, in part, mediated by IGFBP-3 production and theconsequential blockade of IGF action (Buckbinder et al., supra). Theseresults indicate that the IGFBPs can block cell proliferation bymodulating paracrine/autocrine processes regulated by IGF-1/IGF-2. Acorollary to these observations is the finding that prostate-specificantigen (PSA) is an IGFBP-3-protease, which upon activation, increasesthe sensitivity of tumor cells to the actions of IGF-1/IGF-2 due to theproteolytic inactivation of IGFBP-3 (Cohen et al., J. Endocr.,142:407-415 (1994)). The IGFBPs complex with IGF-1/IGF-2 and interferewith the access of IGF-1/IGF-2 to IGF-Rs (Clemmons et al., Anal. NYAcad. Sci. USA, 692:10-21 (1993)). IGFBP-1, -2 and -3 inhibit cellgrowth following addition to cells in vitro (Lee et al., J Endocrinol.,152:39 (1997); Feyen et al., J. Biol. Chem., 266:19469-19474 (1991)).Further, IGFBP-1 (McGuire et al., J. Natl. Cancer Inst.84:1335-1341(1992); Figueroa et al., J Cell Physiol., 157:229-236(1993)), IGFBP-3 (Oh et al. (1995), supra; Pratt and Pollak, Biophys.Res. Commun., 198:292-297 (1994)) and IGFBP-2 have all been shown toinhibit IGF-1 or estrogen-induced breast cancer cell proliferation atnanomolar concentrations in vitro. These findings support the idea thatthe IGFBPs are potent antagonists of IGF action. There is also evidencefor a direct effect of IGFBP-3 on cells through its own cell surfacereceptor, independent of IGF interactions (Oh et al., J. Biol. Chem.,268:14964-14971 (1993); Valentinis et al., Mol. Endocrinol., 9:361-367(1995)). Taken together, these findings underscore the importance of IGFand IGF-R as targets for therapeutic use.

Unlike most other growth factors, the IGFs are present in highconcentrations in the circulation, but only a small fraction of the IGFsis not protein bound. For example, it is generally known that in humansor rodents, less than 1% of the IGFs in blood is in a “free” or unboundform (Juul et al., Clin. Endocrinol., 44: 515-523 (1996); Hizuka et al.,Growth Regulation, 1: 51-55 (1991); Hasegawa et al., J. Clin.Endocrinol. Metab., 80: 3284-3286 (1995)). The overwhelming majority ofthe IGFs in blood circulate as part of a non-covalently associatedternary complex composed of IGF-1 or IGF-2, IGFBP-3, and a large proteintermed the acid-labile subunit (ALS). This complex is composed ofequimolar amounts of each of the three components. The ternary complexof an IGF, IGFBP-3, and ALS has a molecular weight of approximately150,000 daltons, and it has been suggested that the function of thiscomplex in the circulation may be to serve as a reservoir and buffer forIGF-1 and IGF-2, preventing rapid changes in free IGF-1 or IGF-2.

Maintaining normal levels of IGF-1 signaling are important for propercellular function, since both down- and up-regulation of IGF-1-relatedpathways have been implicated in several human diseases. The rate ofcell proliferation is positively correlated with risk of transformationof certain epithelial cell types (Cohen and Ellwein, Science, 249: 1007(1990); Cohen and Ellwein, Cancer Research, 51:6493 (1991)). Relativelyhigh plasma IGF-1 and low IGF binding protein-3 levels are associatedwith greater risk of breast cancer in pre-menopausal women, prostatecancer in men, colorectal cancer in men and women, and lung cancer inmen and women; additional in vitro and in vivo studies reflecting a linkbetween IGF and cancer are found in “Insulin-Like Growth Factors andCancer”, Cytokine Bulletin, R&D Systems (Fall 2000 edition), pages 2-3.IGFs have mitogenic and anti-apoptotic influences on normal andtransformed prostate epithelial cells (Hsing et al., Cancer Research,56: 5146 (1996); Culig et al., Cancer Research, 54: 5474 (1994); Cohenet al., Hormone and Metabolic Research, 26: 81 (1994); Iwamura et al.,Prostate, 22: 243 (1993); Cohen et al., J. Clin. Endocrin. & Metabol.,73: 401 (1991); Rajah et al., J. Biol. Chem., 272: 12181 (1997)). Mostcirculating IGF-1 originates in the liver, but IGF bioactivity intissues is related not only to levels of circulating IGFs and IGFBPs,but also to local production of IGFs, IGFBPs, and IGFBP proteases (Jonesand Clemmons, Endocrine Reviews, 16: 3 (1995)). Person-to-personvariability in levels of circulating IGF-1 and IGFBP-3 (the majorcirculating IGFBP (Jones and Clemmons, supra)) is considerable (Juul etal., J. Clin. Endocrinol. & Metabol., 78: 744 (1994); Juul et al, J.Clin. Endocrinol. & Metabol., 80: 2534 (1995)), and heterogeneity inserum IGF-1 level appears to reflect heterogeneity in tissue IGFbioactivity. Markers relating to IGF-axis components can be used as arisk marker for prostate cancer, as PSA is likewise used (WO 99/38011).Further, it has been found that reduced IGF-1 concentrations in serumcorrelate with improved clinical scores in acromegaly patients (Traineret al., New England J. Med., 342: 1171-1177 (2000)).

There has been much work identifying the regions on IGF-1 and IGF-2 thatbind to the IGFBPs (Bayne et al., J. Biol. Chem., 265: 15648-15652(1990); Dubaquie and Lowman, Biochemistry, 38: 6386-6396 (1999); andU.S. Pat. Nos. 5,077,276; 5,164,370; and 5,470,828). For example, it hasbeen discovered that the N-terminal region of IGF-1 and IGF-2 iscritical for binding to the IGFBPs (U.S. Pat. Nos. 5,077,276; 5,164,370;and 5,470,828). Thus, the natural IGF-1 variant, designated des (1-3)IGF-1, binds poorly to IGFBPs.

A similar amount of research has been devoted to identifying the regionson IGF-1 and IGF-2 that bind to IGF-1R (Bayne et al., supra; Oh et al.,Endocrinology (1993), supra). It was found that the tyrosine residues inIGF-1 at positions 24, 31, and 60 are crucial to the binding of IGF-1 toIGF-1R (Bayne et al., supra). Mutant IGF-1 molecules where one or moreof these tyrosine residues are substituted showed progressively reducedbinding to IGF-1R. Bayne et al., supra, also investigated whether suchmutants of IGF-1 could bind to IGF-1R and to the IGFBPs. They found thatquite different residues on IGF-1 and IGF-2 are used to bind to theIGFBPs from those used to bind to IGF-R. It is therefore possible toproduce IGF variants that show reduced binding to the IGFBPs, but,because they bind well to IGF-1R, show maintained activity in in vitroactivity assays.

Also reported was an IGF variant that binds to IGFBPs but not to IGFreceptors and therefore shows reduced activity in in vitro activityassays (Bar et al., Endocrinology, 127: 3243-3245 (1990)). In thisvariant, designated (1-27,gly⁴, 38-70)-hIGF-1, residues 28-37 of the Cregion of human IGF-1 are replaced by a four-residue glycine bridge.

Other truncated IGF-1 variants are disclosed. For example, in the patentliterature, WO 96/33216 describes a truncated variant having residues1-69 of authentic IGF-1. EP 742,228 discloses two-chain IGF-1superagonists, which are derivatives of the naturally occurring,single-chain IGF-1 having an abbreviated C region. The IGF-1 analogs areof the formula: BC^(n),A

wherein B is the B region of IGF-1 or a functional analog thereof, C isthe C region of IGF-1 or a functional analog thereof, n is the number ofamino acids in the C region and is from about 6 to about 12, and A isthe A region of IGF-1 or a functional analog thereof.

Additionally, Cascieri et al., Biochemistry, 27: 3229-3233 (1988)discloses four mutants of IGF-1, three of which have reduced affinity toIGF-1R. These mutants are: (Phe²³1,Phe²⁴,Tyr²⁵)IGF-1 (which isequipotent to human IGF-1 in its affinity to the Types 1 and 2 IGF andinsulin receptors), (Leu²⁴)IGF-1 and (Ser²⁴)IGF-1 (which have a loweraffinity than IGF-1 to the human placental IGF-1R, the placental insulinreceptor, and the IGF-1R of rat and mouse cells), and desoctapeptide(Leu²⁴)IGF-1 (in which the loss of aromaticity at position 24 iscombined with the deletion of the carboxyl-terminal D region of hIGF-1,which has lower affinity than (Leu²⁴)IGF-1 for the IGF-1R and higheraffinity for the insulin receptor). These four mutants have normalaffinities for human serum binding proteins.

Bayne et al., J. Biol. Chem., 263: 6233-6239 (1988) discloses fourstructural analogs of human IGF-1: a B-chain mutant in which the first16 amino acids of IGF-1 were replaced with the first 17 amino acids ofthe B-chain of insulin, (Gln³,Ala⁴)IGF-1, (Tyr¹⁵,Leu⁶)IGF-1, and(Gln³,Ala⁴,Tyr¹⁵,Leu⁶)IGF-1. These studies identify some of the regionsof IGF-1 that are responsible for maintaining high-affinity binding withthe serum binding protein and the Type 2 IGF receptor.

In another study, Bayne et al., J. Biol. Chem., 264: 11004-11008 (1988)discloses three structural analogs of IGF-1: (1-62)IGF-1, which lacksthe carboxyl-terminal 8-amino-acid D region of IGF-1;(1-27,Gly⁴,38-70)IGF-1, in which residues 28-37 of the C region of IGF-1are replaced by a four-residue glycine bridge; and(1-27,Gly⁴,38-62)IGF-1, with a C region glycine replacement and a Dregion deletion. Peterkofsky et al., Endocrinology, 128: 1769-1779(1991) discloses data using the Gly⁴ mutant of Bayne et al., supra (vol.264).

Cascieri et al., J. Biol. Chem., 264: 2199-2202 (1989) discloses threeIGF-1 analogs in which specific residues in the A region of IGF-1 arereplaced with the corresponding residues in the A chain of insulin. Theanalogs are:(Ile⁴¹,Glu⁴⁵,Gln⁴⁶,Thr⁴⁹,Ser⁵⁰,Ile⁵¹,Ser⁵³,Tyr⁵⁵,Gln⁵⁶)IGF-1, an A-chainmutant in which residue 41 is changed from threonine to isoleucine andresidues 42-56 of the A region are replaced; (Thr⁴⁹,Ser⁵⁰,Ile⁵)IGF-1;and (Tyr⁵⁵,Gln⁵⁶)IGF-1.

Clemmons et al., J. Biol. Chem., 265: 12210-12216 (1990) discloses useof IGF-1 analogs that have reduced binding affinity for either IGF-1R orbinding proteins to study the ligand specificity of IGFBP-1 and the roleof IGFBP-1 in modulating the biological activity of IGF-1.

WO 94/04569 discloses a specific binding molecule, other than a naturalIGFBP, that is capable of binding to IGF-1 and can enhance thebiological activity of IGF-1.

The direction of research into IGF variants has mostly been to make IGFvariants that do not bind to the IGFBPs, but show maintained binding tothe IGF receptor. The idea behind the study of such molecules is thatthe major actions of the IGFBPs are proposed to be an inhibition of theactivity of the IGFs. Chief among these variants is the naturalmolecule, des(1-3)IGF-1, which shows selectively reduced affinity forsome of the IGF binding proteins, yet a maintained affinity for the IGFreceptor (U.S. Pat. Nos. 5,077,276; 5,164,370; 5,470,828).

Peptides that bind to IGFBP-1, block IGF-1 binding to this bindingprotein, and thereby release “free-IGF” activity from mixtures of IGF-1and IGFBP-1 have been recently described (Lowman et al., Biochemistry,37: 8870-8878 (1998); WO 98/45427 published Oct. 15, 1998; Lowman etal., International Pediatric Nephrology Association, Fifth Symposium onGrowth and Development in Children with Chronic Renal Failure (New York,Mar. 13, 1999)).

Exploitation of the interaction between IGF and IGFBP in screening,preventing, or treating disease has been limited, however, because of alack of specific antagonists. To date, only one publication is known toexist that describes the application of an IGF-1/IGF-2 antagonist as apotential therapeutic adjunct in the treatment of cancer (Pietrzkowskiet al., Cancer Res., 52: 6447-6451 (1992)). In that report, a peptidecorresponding to the D-region of IGF-1 was synthesized for use as anIGF-1/2 antagonist. This peptide exhibited questionable inhibitoryactivity against IGF-1. The basis for the observed inhibition is unclearas the D-region does not play a significant role in IGF-1R binding butrather, in IGF-1 binding to the insulin receptor (Cooke et al.,Biochem., 30:5484-5491 (1991); Bayne et al., J. Biol. Chem.,264:11004-11008 (1988); Yee et al., Cell Growth and Different., 5:73-77(1994)). IGF antagonists whose mechanism of action is via blockade ofinteractions at the IGF-1R interface may also significantly alterinsulin action at the insulin receptor, a disadvantage of suchantagonists.

Recently, certain IGF-1 antagonists have been described by WO 00/23469,which discloses the portions of IGFBP and IGF peptides that account forIGF-IGFBP binding, i.e., an isolated IGF binding domain of an IGFBP ormodification thereof that binds IGF with at least about the same bindingaffinity as the full-length IGFBP. The patent publication also disclosesan IGF antagonist that reduces binding of IGF to an IGF receptor, and/orbinds to a binding domain of IGFBP. Disclosed uses of such antagonistsand fragments are in treating a subject having cancer and preventingcancer in a subject, treating a subject with a diabetic complicationexacerbated by IGF and preventing diabetic complications exacerbated byIGF, or treating a subject with an ischemic injury or preventing anischemic injury in a subject.

Additionally, EP 639981 discloses pharmaceutical compositions comprisingshort peptides that function as IGF-1 receptor antagonists. The peptidesused in the pharmaceutical compositions consist of less than 25 aminoacids, comprise at least a portion of the C or D region from IGF-1, andinhibit IGF-1-induced autophosphorylation of IGF-1 receptors. Methods ofinhibiting cell proliferation and of treating individuals suspected ofsuffering from or susceptible to diseases associated with undesirablecell proliferation such as cancer, restenosis and asthma are disclosed.

Generation of specific IGF-1 antagonists has been restricted, at leastin part, because of difficulties in studying the structure of IGF andIGFBP. Due to the inability to obtain crystals of IGF-1 suitable fordiffraction studies, for example, an extrapolation of IGF-1 structurebased on the crystal structure of porcine insulin was the most importantstructural road map for IGF-1 available (Blundell et al., Proc. Natl.Acad. Sci. USA, 75:180-184 (1978)). See also Blundell et al., Fed. Proc.42: 2592 (1983), which discloses tertiary structures, receptor binding,and antigenicity of IGFs. Based on studies of chemically modified andmutated IGF-1, a number of common residues between IGF-1 and insulinhave been identified as being part of the IGF-1R-insulin receptorcontact site, in particular the aromatic residues at positions 23-25.Using NMR and restrained molecular dynamics, the solution structure ofIGF-1 was recently reported (Cooke et al., supra). The resultingminimized structure was shown to better fit the experimental findings onmodified IGF-1, as well as the extrapolations made from thestructure-activity studies of insulin. Further, De Wolf et al., ProteinSci., 5: 2193 (1996) discloses the solution structure of a mini-IGF-1.Sato et al., Int. J. Pept., 41: 433 (1993) discloses thethree-dimensional structure of IGF-1 determined by 1H-NMR and distancegeometry. Torres et al., J Mol Biol. 248: 385 (1995) discloses thesolution structure of human IGF-2 and its relationship to receptor andbinding protein interactions. Laajoki et al., J. Biol. Chem., 275: 10009(2000) discloses the solution structure and backbone dynamics oflong-[Arg(3)]IGF-1.

Peptide sequences capable of binding to insulin and/or insulin-likegrowth factor receptors with either agonist or antagonist activity andidentified from various peptide libraries are described in WO 01/72771published Oct. 4, 2001.

There is a continuing need in the art for a molecule that acts as an IGFantagonist to control the levels of circulating IGF as well as receptorresponse, for therapeutic or diagnostic purposes.

SUMMARY OF THE INVENTION

Accordingly, the invention is as claimed. In one aspect the inventionprovides a peptide of family 1 comprising the sequence:(Xaa)₁(Xaa)₂Cys(Xaa)₃(Xaa)₄SerVal(Xaa)₅AlaLeu(Xaa)₆(Xaa)₇CysMet(Xaa)₈(SEQ ID NO:1) where (Xaa)₁, (Xaa)₂, and (Xaa)₇ are any amino acid,(Xaa)₃ is Phe, Leu, or Tyr, (Xaa)₄ is Glu, Asp, Ala, Gly, Thr, or Ser,(Xaa)₅ is Glu, Asp, Ala, or Gly, (Xaa)₆ is Arg or Lys, and (Xaa)₈ is Tyror Arg. (Xaa)₄ is Glu, Ala, Gly, Thr, or Ser, (Xaa)₅ is Glu, Ala, orGly, and (Xaa)₈ is Tyr. The preferred peptides of the above sequence aresuch that (Xaa)₄ is Glu, Ala or Thr, (Xaa)₅ is Ala or Gly, and Xaa8 isTyr. More preferred are the peptides wherein (Xaa)₄ is Glu or Ala,(Xaa)₅ is Ala or Gly, and (Xaa)₈ is Tyr. Still more preferred are thepeptides comprising the sequence RNCFESVAALRRCMYG (SEQ ID NO:2),MDCLASVEALKWCMYG (SEQ ID NO:3), or FECLTSVEALRGCMYG (SEQ ID NO:4). Mostpreferred are peptides that comprise SEQ ID NO:2 or 3.

In another aspect, the invention provides a peptide of family 2comprising the sequence: (Xaa)₁(Xaa)₂Cys(Xaa)₃(Xaa)₄Asp(Xaa)₅(Xaa)₆Gly(Xaa)₇(Xaa)₈TyrCysTrp(Xaa)₉ (SEQID NO:5), where (Xaa)₁, (Xaa)₄, and (Xaa)₈ are any amino acid, (Xaa)₂ isArg, Lys, Gly, Ser, or Thr, (Xaa)₃ is Ala or Val, (Xaa)₅ is Ala or Leu,(Xaa)₆ is Ala, Gly, or Leu, (Xaa)₇ is Phe, Tyr, Trp, or Gly, and (Xaa)₉is Glu, Asp, Ala, or Gly. The preferred peptides herein are such that(Xaa)₂ is Gly, Ser, Arg, or Thr, and (Xaa)₉ is Glu, Ala, or Asp. Morepreferred are peptides wherein (Xaa)₂ is Glu or Arg, (Xaa)₅ is Leu,(Xaa)₆ is Ala or Gly, (Xaa)₇ is Phe, and (Xaa)₉ is Ala. The mostpreferred of this family of peptides are those that comprise thesequence LGCASDLAGFWYCWAG (SEQ ID NO:6) or WRCVDDLGGFQYCWAG (SEQ IDNO:7).

Preferably, all the amino acids in these two families of peptides areL-amino acids. Also preferred is that these families of peptidescomprise a glycine residue after (Xaa)₈ for family 1 above or after(Xaa)₉ for family 2 above.

The invention also provides conjugates comprising the peptide conjugatedwith a cytotoxic agent or polyethylene glycol. The cytotoxic agent heremay be one that is active in killing cells once internalized.

Uses of these peptides include all uses that antagonize at least onebiological activity of exogenous or endogenous IGFs. They can be used intreating, inhibiting, or preventing conditions in which an IGFantagonist such as IGFBP-3 or antibodies to IGF-1 is useful, asdescribed below.

The invention also provides a composition comprising one of the peptidesdescribed above in a carrier. Preferably, this composition is sterileand the carrier is a pharmaceutically acceptable carrier. Also preferredis the composition further comprising an angiogenic agent orchemotherapeutic agent, and also one that is suitable for injection orinhalation. A kit is also provided comprising a container containing thecomposition and instructions directing the user to utilize thecomposition.

In another aspect, the invention provides a method for treating a mammalhaving a disorder involving an IGF-1-mediated event comprisingadministering to the mammal an effective amount of any of the peptidesor compositions described above. More specifically, the inventionprovides a method of treating a mammal suffering from, or predisposedto, a disease or disorder involving an IGF-1-mediated event, comprisingadministering to the mammal a therapeutically effective amount of apeptide as disclosed herein, or of a composition comprising the peptideand a pharmaceutically acceptable carrier. Preferably, this methodfurther comprises administering to the mammal an effective amount ofanother agent that treats said disorder. This agent may be a growthinhibitory agent, an angiostatic agent, or a cytotoxic agent, or achemotherapeutic agent or an antibody. In another preferred aspect, themammal is human.

In a further preferred embodiment, before the administration step of theabove method, the concentration of IGF-1 in a body sample from themammal is measured, wherein an elevated concentration of IGF-1 above areference range for IGF-1 indicates an increased risk for the disorder.The body sample is preferably selected from the group consisting oftumor tissue, blood, plasma, serum, mammary fluid, and seminal fluid. Inanother preferred embodiment, the IGF-1 is total IGF-1, free IGF-1 orcomplexed IGF-1, and the disorder is cancer, a diabetic complicationexacerbated by IGF-1, preferably diabetic retinopathy or diabeticnephropathy, acromegaly, age-related macular degeneration, ischemicinjury, or a trauma.

If the disorder is cancer, preferably it comprises a tumor thatexpresses an insulin-like growth factor receptor. Further, the cancer ispreferably breast cancer, prostate cancer, colorectal cancer, or lungcancer, more preferably breast or prostate cancer. If the disorder isprostate cancer the process preferably comprises, before theadministration step, measuring the concentration of PSA in a body samplefrom the mammal, wherein an elevated concentration of PSA above areference range for PSA indicates an increased risk for prostate cancer.Alternatively, if the disorder is prostate cancer, the method preferablycomprises, before the administration step, measuring the concentrationof IGF-1 in a body sample from the mammal, measuring the concentrationof IGFBP-3 in a body sample from the mammal and conducting amultivariate adjustment of the IGF-1 concentration relative to theIGFBP-3 concentration to provide an adjusted IGF-1 level, wherein theadjusted IGF-1 level above a reference range for adjusted IGF-1indicates an increased risk for prostate cancer. Still alternatively, ifthe disorder is prostate cancer, the method preferably comprises, beforethe administration step, measuring the concentration of IGF-1 in a bodysample from the mammal, measuring the concentration of IGFBP-3 in a bodysample from the mammal, measuring the concentration of PSA in a bodysample from the mammal, and conducting a multivariate adjustment of theIGF-1 concentration relative to the IGFBP-3 concentration and PSAconcentration to provide an adjusted IGF/IGFBP/PSA value, wherein anadjusted IGF/IGFBP/PSA value above a reference range for adjustedIGF/IGFBP/PSA indicates an increased risk for severe prostate cancer.

The present invention further provides various dosage forms of any ofthe peptides of the present invention, including but not limited to,those suitable for parenteral, oral, rectal and pulmonary administrationof a peptide. In preferred aspects herein a therapeutic dosage form isprovided suitable for inhalation and the invention provides for thetherapeutic treatment of diseases or disorders involving an IGF-mediatedor associated process or event via pulmonary administration of a peptideof the invention. More particularly, the invention is directed topulmonary administration of the peptides herein by inhalation. Thus, thepresent invention provides an aerosol formulation comprising an amountof a peptide of the invention, effective to block or prevent anIGF-mediated or associated process or event and a dispersant. In oneembodiment, any one of the above peptides can be provided in a liquidaerosol formulation. Alternatively, the peptide can be provided as a drypowder aerosol formulation. Therefore, according to the presentinvention, formulations are provided that provide an effectivenon-invasive alternative to other parenteral routes of administration ofthe peptides herein for the treatment of IGF-mediated or associatedevents.

Isolated nucleic acid encoding one of the above peptides herein is alsoprovided, and may be used for in vivo or ex vivo gene therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the three-dimensional minimized mean structure of thepeptide IGF-F1-1 in solution calculated using restraints derived fromNMR data. The backbone fold is depicted as a ribbon, and allside-chains' heavy atoms are shown; several side-chains are labeled.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Definitions

As used herein, “mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic, and farm animals,and zoo, sports, or pet animals, such as dogs, horses, cats, sheep,pigs, cows, etc. The preferred mammal herein is a human. The term“non-adult” refers to mammals that are from perinatal age (such aslow-birth-weight infants) up to the age of puberty, the latter beingthose that have not yet reached full growth potential.

As used herein, “IGF” refers to native insulin-like growth factor-1 andnative insulin-like growth factor-2 as well as natural variants thereofsuch as brain IGF, otherwise known as des(1-3)IGF-1.

As used herein, “IGF-1” refers to insulin-like growth factor-1 from anyspecies, including bovine, ovine, porcine, equine, and human, preferablyhuman, and, if referring to exogenous administration, from any source,whether natural, synthetic, or recombinant. Human native-sequence,mature IGF-1, more preferably without a N-terminal methionine isprepared, e.g., by the process described in EP 230,869 published Aug. 5,1987; EP 128,733 published Dec. 19, 1984; or EP 288,451 published Oct.26, 1988. More preferably, this native-sequence IGF-1 is recombinantlyproduced and is available from Genentech, Inc., South San Francisco,Calif. for clinical investigations.

As used herein, “IGF-2” refers to insulin-like growth factor-2 from anyspecies, including bovine, ovine, porcine, equine, and human, preferablyhuman, and, if referring to exogenous administration, from any source,whether natural, synthetic, or recombinant. It may be prepared by themethod described in, e.g., EP 128,733, supra.

An “IGFBP” or an “IGF binding protein” refers to a protein orpolypeptide normally associated with or bound or complexed to IGF-1 orIGF-2, whether or not it is circulatory (i.e., in serum or tissue). Suchbinding proteins do not include receptors. This definition includesIGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, Mac 25 (IGFBP-7),and prostacyclin-stimulating factor (PSF) or endothelial cell-specificmolecule (ESM-1), as well as other proteins with high homology toIGFBPs. Mac 25 is described, for example, in Swisshelm et al., Proc.Natl. Acad. Sci. USA, 92: 4472-4476 (1995) and Oh et al., J. Biol.Chem., 271: 30322-30325 (1996). PSF is described in Yamauchi et al.,Biochemical Journal, 303: 591-598 (1994). ESM-1 is described in Lassalleet al., J. Biol. Chem., 271: 20458-20464 (1996). For other identifiedIGFBPs, see, e.g., EP 375,438 published 27 Jun. 1990; EP 369,943published 23 May 1990; WO 89/09268 published 5 Oct. 1989; Wood et al.,Molecular Endocrinology, 2: 1176-1185 (1988); Brinkman et al., The EMBOJ., 7: 2417-2423 (1988); Lee et al., Mol. Endocrinol., 2: 404-411(1988); Brewer et al., BBRC, 152: 1289-1297 (1988); EP 294,021 published7 Dec. 1988; Baxter et al., BBRC, 147: 408-415 (1987); Leung et al.,Nature, 330: 537-543 (1987); Martin et al., J. Biol. Chem., 261:8754-8760 (1986); Baxter et al., Comp. Biochem. Physiol., 91B: 229-235(1988); WO 89/08667 published 21 Sep. 1989; WO 89/09792 published 19Oct. 1989; and Binkert et al., EMBO J., 8: 2497-2502 (1989).

The term “body sample” refers to a biological specimen from a mammal,preferably from a human, including tissues, cells, and body fluid.Examples include tumor tissue, tumor cells, serum, plasma, lymph fluid,synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk,mammary fluid, whole blood, urine, spinal fluid, saliva, sputum, tears,perspiration, mucus, tissue culture medium, tissue extracts, andcellular extracts. Preferably, the body sample is tumor tissue, blood,plasma, serum, mammary fluid, or seminal fluid.

As used herein, “human IGF receptor” refers to any receptor for an IGFfound in humans and includes the Type 1 and Type 2 IGF receptors inhumans to which both human IGF-1 and IGF-2 bind, such as the placentalIGF-1R, etc.

The term “amino acid” within the scope of the present invention is usedin its broadest sense and is meant to include the naturally-occurring L∀-amino acids or residues. The commonly used one- and three-letterabbreviations for naturally-occurring amino acids are used herein(Lehninger, Biochemistry, 2d ed., pp. 71-92, (Worth Publishers: NewYork, 1975). The term includes D-amino acids as well aschemically-modified amino acids such as amino acid analogs,naturally-occurring amino acids that are not usually incorporated intoproteins such as norleucine, and chemically-synthesized compounds havingproperties known in the art to be characteristic of an amino acid. Forexample, analogs or mimetics of phenylalanine or proline, which allowthe same conformational restriction of the peptide compounds as naturalPhe or Pro, are included within the definition of amino acid. Suchanalogs and mimetics are referred to herein as “functional equivalents”of an amino acid. Other examples of amino acids are listed by Robertsand Vellaccio, The Peptides: Analysis, Synthesis, Biology, Eds. Grossand Meiehofer, Vol. 5, p. 341 (Academic Press, Inc.: N.Y. 1983).

The term “conservative” amino acid substitution as used herein to referto amino acid substitutions that substitute functionally-equivalentamino acids. Conservative amino acid changes result in silent changes inthe amino acid sequence of the resulting peptide. For example, one ormore amino acids of a similar polarity act as functional equivalents andresult in a silent alteration within the amino acid sequence of thepeptide. The largest sets of conservative amino acid substitutionsinclude:

(1) hydrophobic: His, Trp, Tyr, Phe, Met, Leu, Ile, Val, Ala;

(2) neutral hydrophilic: Cys, Ser, Thr;

(3) polar: Ser, Thr, Asn, Gln; (4) acidic/negatively charged: Asp, Glu;

(5) charged: Asp, Glu, Arg, Lys, His;

(6) basic/positively charged: Arg, Lys, His;

(7) basic: Asn, Gln, His, Lys, Arg;

(8) residues that influence chain orientation: Gly, Pro; and

(9) aromatic: Trp, Tyr, Phe, His.

In addition, “structurally-similar” amino acids can substituteconservatively for some of the specific amino acids. Groups ofstructurally-similar amino acids include: (Ile, Leu, and Val); (Phe andTyr); (Lys and Arg); (Gln and Asn); (Asp and Glu); and (Gly and Ala). Inthis regard, it is understood that amino acids are substituted on thebasis of side-chain bulk, charge, and/or hydrophobicity. Amino acidresidues are classified into four major groups:

Acidic: The residue has a negative charge due to loss of an H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous solution.

Basic: The residue has a positive charge due to association with an Hion at physiological pH and the residue is attracted by aqueous solutionso as to seek the surface positions in the conformation of a peptide inwhich it is contained when the peptide is in aqueous medium atphysiological pH.

Neutral/non-polar: The residues are not charged at physiological pH andthe residue is repelled by aqueous solution so as to seek the innerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium. These residues are also designated“hydrophobic residues.”

Neutral/polar: The residues are not charged at physiological pH, but theresidue is attracted by aqueous solution so as to seek the outerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium.

“Amino acid” residues can be further classified as cyclic or non-cyclic,and aromatic or non-aromatic with respect to their side-chain groups,these designations being commonplace to the skilled artisan. The tablebelow shows the types of conservative substitutions that can be made.Original Exemplary Conservative Preferred Conservative ResidueSubstitution Substitution Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Gln, His, Lys, Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn Asn Glu AspAsp Gly Pro Pro His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala LeuPhe Leu Ile, Val Ile Met, Ala, Phe Lys Arg, Gln, Asn Arg Met Leu, Phe,Ile Leu Phe Leu, Val, Ile, Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser SerTrp Tyr Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe Leu Ala

Peptides synthesized by the standard solid-phase synthesis techniquesdescribed herein, for example, are not limited to amino acids encoded bygenes for substitutions involving the amino acids. Commonly-encounteredamino acids that are not encoded by the genetic code include, forexample, those described in WO 90/01940 and in the table below, as wellas, for example, 2-amino adipic acid (Aad) for Glu and Asp;2-aminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric (Abu) acid forMet, Leu, and other aliphatic amino acids; 2-aminoheptanoic acid (Ahe)for Met, Leu, and other aliphatic amino acids; 2-aminoisobutyric acid(Aib) for Gly; cyclohexylalanine (Cha) for Val, Leu and Ile;homoarginine (Har) for Arg and Lys; 2,3-diaminopropionic acid (Dpr) forLys, Arg, and His; N-ethylglycine (EtGly) for Gly, Pro, and Ala;N-ethylglycine (EtGly) for Gly, Pro, and Ala; N-ethylasparagine (EtAsn)for Asn, and Gln; hydroxylysine (Hyl) for Lys; allohydroxylysine (AHyl)for Lys; 3-(and 4-)hydroxyproline (3Hyp, 4Hyp) for Pro, Ser, and Thr;allo-isoleucine (AIle) for Ile, Leu, and Val; Δ-amidinophenylalanine forAla; N-methylglycine (MeGly, sarcosine) for Gly, Pro, and Ala;N-methylisoleucine (MeIle) for Ile; norvaline (Nva) for Met and otheraliphatic amino acids; norleucine (Nle) for Met and other aliphaticamino acids; ornithine (Orn) for Lys, Arg and His; citrulline (Cit) andmethionine sulfoxide (MSO) for Thr, Asn, and Gln; andN-methylphenylalanine (MePhe), trimethylphenylalanine, halo-(F—, Cl—,Br—, or I—)phenylalanine, or trifluorylphenylalanine for Phe.Abbreviations used in the specification Compound Abbreviation Acetyl AcAlanine Ala 3-(2-Thiazolyl)-L-alanine Tza Arginine Arg Asparagine AsnAspartic acid Asp t-Butyloxycarbonyl BocBenzotriazol-1-yloxy-tris-(dimethylamino) Bopphosphonium-hexafluorophosphate ∃-Alanine ∃Ala ∃-Valine ∃Val∃-(2-Pyridyl)-alanine Pal(2) ∃-(3-Pyridyl)-alanine Pal(3)∃-(4-Pyridyl)-alanine Pal(4) ∃-(3-N-Methylpyridinium)-alanine PalMe(3)t-Butyl tBu, But t-Butyloxycarbonyl Boc Caffeic acid Caff Cysteine CysCyclohexylalanine Cha Cyclohexylglycine Chg 3,5-DinitrotyrosineTyr(3,5-No₂) 3,5-Diiodotyrosine Tyr(3,5-I) 3,5-DibromotyrosineTyr(3,5-Br) 9-Fluorenylmethyloxy-carbonyl Fmoc Glutamine Gln Glutamicacid Glu (-Carboxyglutamic acid Gla Glycine Gly Histidine HisHomoarginine hArg 3-Hydroxyproline Hyp Isoleucine Ile Leucine Leutert-Leucine Tle Lysine Lys Mercapto-∃,∃-cyclopentamethylene- Mpppropionic acid Mercaptoacetic acid Mpa Mercaptopropionic acid MprMethionine Met 1-Naphthylalanine Nal(1) 2-Naphthylalanine Nal(2)Nicotinic acid Nic Nipecotic acid Npa N-methyl nicotinic acid NicMeNorarginine nArg Norleucine Nle Norvaline Nva Ornithine OrnOrnithine-derived dimethylamidinium Orn(N*—C₃H₇N) Phenylalanine Phep-Guanidinophenylalanine Phe(Gua) p-Aminophenylalanine Phe(NH₂)p-Chlorophenylalanine Phe(Cl) p-Flurophenylalanine Phe(F)p-Nitrophenylalanine Phe(NO₂) p-Hydroxyphenylglycine Pgl(OH)p-Toluenesulfonyl Tos m-Amidinophenylalanine mAph p-AmidinophenylalaninepAph Phenylglycine Pgl Phenylmalonic acid Pma Proline Pro4-Quinolinecarboxy 4-Qca Sarcosine Sar Serine Ser Threonine ThrTryptophan Trp Tyrosine Tyr 3-iodotyrosine Tyr(3-I) O-Methyl tyrosineTyr(Me) Valine Val*Amino acids of D configuration are denoted by D-prefix usingthree-letter code (e.g, D-Ala, D-Cys, D-Asp, D-Trp).

“Peptides” include molecules having at least two amino acids and includepolypeptides having at least about 60 amino acids. Preferably, thepeptides have about 10 to about 60 amino acids, more preferably about10-25, and most preferably about 12-25 amino acids. The definitionincludes linear and cyclic peptides, peptide derivatives, their salts,or optical isomers.

As used herein, an “amide bond-forming substituent contained in an aminoacid side-chain”, a “side-chain amide bond-forming substituent”, andtheir grammatical variants, are defined to include (1) any carboxysubstituent contained in the side-chain (“R” group) of an amino acidwherein the carboxy substituent is capable of forming an amide linkagewith an amino group contained in another molecule, i.e., the carboxysubstituent reacts with an amino group contained in another molecule toform an amide linkage; and (2) any amino substituent contained in theside-chain (“R” group) of an amino acid wherein the amino substituent iscapable of forming an amide linkage with a carboxy group contained inanother molecule, i.e., the amino substituent reacts with a carboxygroup contained in another molecule to form an amide linkage.

As used herein, “differentially-removable” protecting or protectivegroups are defined as any pair of protective groups capable ofprotecting a first amide bond-forming substituent and a second amidebond-forming substituent, wherein it is possible to deprotect the firstamide bond-forming substituent protected with one member of the pairunder conditions which do not deprotect the second amide bond-formingsubstituent protected with the other member of the pair.Differentially-removable protecting groups are also referred to hereinas “orthogonal” protecting groups, and the differentially-removableprotection conferred by such protective groups is referred to herein as“orthogonal” protection.

As used herein, the term “treating” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those prone to havingthe disorder or diagnosed with the disorder or those in which thedisorder is to be prevented. Consecutive treatment or administrationrefers to treatment on at least a daily basis without interruption intreatment by one or more days. Intermittent treatment or administration,or treatment or administration in an intermittent fashion, refers totreatment that is not consecutive, but rather cyclic in nature. Thetreatment regime herein can be either consecutive or intermittent.Subjects for whom the preventive measures are appropriate include thosewith one or more known risk factors for the disorder, such as cancer.

As used herein, the term “pulmonary administration” refers toadministration of a formulation of the invention through the lungs byinhalation. As used herein, the term “inhalation” refers to intake ofair to the alveoli. In specific examples, intake can occur byself-administration of a formulation of the invention while inhalingthrough a nebulizer or other aerosol-delivery device, or byadministration via a respirator, e.g., to a patient on a respirator. Theterm “inhalation” used with respect to a formulation of the invention issynonymous with “pulmonary administration.”

As used herein, the term “parenteral” refers to introduction of apeptide of the invention into the body by other than the intestines, andin particular, intravenous (i.v.), intraarterial (i.a.), intraperitoneal(i.p.), intramuscular (i.m.), intraventricular, and subcutaneous (s.c.)routes.

As used herein, the term “aerosol” refers to suspension in the air. Inparticular, aerosol refers to the formation of particles or particulatesin a formulation of the invention and its suspension in the air.According to the present invention, an aerosol formulation is aformulation comprising a peptide of the present invention that issuitable for aerosolization, i.e., formation of particles orparticulates and suspension in the air, for inhalation or pulmonaryadministration.

As used herein, the term “dispersant” refers to an agent that assistsaerosolization of the peptide or absorption of the protein in lungtissue, or both. Preferably, the dispersant is pharmaceuticallyacceptable. As used herein, the modifier “pharmaceutically-acceptable”means approved by a regulatory agency of the federal or a stategovernment or listed in the U.S. Pharmacopoeia or other generallyrecognized pharmacopoeia for use in animals, and more particularly inhumans.

A “disorder” is any condition caused, mediated, or exacerbated by, orassociated with, an IGF, preferably IGF-1, that would benefit fromtreatment with the peptides herein. This includes chronic and acutedisorders or diseases including those pathological conditions thatpredispose the mammal to the disorder in question. Non-limiting examplesof disorders to be treated herein include diseases associated withundesirable cell proliferation, such as benign tumors, cancer,restenosis, and asthma; acromegaly; inflammatory, angiogenic, orimmunological disorders; an ischemic injury such as a stroke, myocardialischemia, or ischemic injury to the kidneys; diabetic complications suchas diabetic retinopathies or neuropathies; eye-related diseases; orneuronal, glial, astrocytal, hypothalamic or other glandular,macrophagal, epithelial, stromal, or blastocoelic disorders. Eye-relateddisorders include age-related macular degeneration; ophthalmic surgerysuch as cataract extraction, corneal transplantation, glaucomafiltration surgery, and keratoplasty; surgery to correct refraction,i.e., a radial keratotomy, also in sclera macular holes anddegeneration; retinal tears; vitreoretinopathy; cataract disorders ofthe cornea such as the sequelae of radial keratotomy; dry eye; viralconjunctivitis; ulcerative conjunctivitis; optical wounds such ascorneal epithelial wounds; Sjogren's syndrome; macular and retinaledema; vision-limited scarring; and retinal ischemia. Preferably, suchdisorders are cancer, a diabetic complication, an ischemic injury,acromegaly, restenosis, an eye-related disorder, or asthma. The efficacyof the treatment can be evidenced by a reduction in clinicalmanifestations or symptoms, including, for example, decreased cellproliferation or growth, improved renal clearance, improved vision, or areduction in the amount of IGF available for binding to the IGFreceptor.

The term “effective amount” refers to an amount of a peptide effectiveto treat a disease or disorder in a mammal. In the case of cancer, theeffective amount of the peptide may reduce the number of cancer cells;reduce the tumor size; inhibit (i.e., slow to some extent and preferablystop) cancer cell infiltration into peripheral organs; inhibit (i.e.,slow to some extent and preferably stop) tumor metastasis; inhibit, tosome extent, tumor growth; and/or relieve to some extent one or more ofthe symptoms associated with the disorder. To the extent the peptide mayprevent growth and/or kill existing cancer cells, it may be cytostaticand/or cytotoxic. For cancer therapy, efficacy in vivo can, for example,be measured by assessing the time to disease progression (TTP) and/ordetermining the response rates (RR).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer, lungcancer (including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD).Preferably, the cancer comprises a tumor that expresses an IGF receptor,more preferably breast cancer, lung cancer, colorectal cancer, orprostate cancer, and most preferably breast or prostate cancer.

An “another agent that treats the disorder” is any agent other than thepeptides herein that in effective amounts will treat the disorder inquestion. This includes a growth inhibitory agent, an angiostatic agent,or a cytotoxic agent. Preferably, the agent is a chemotherapeutic agentor antibody, preferably a growth-inhibitory antibody, an antibody thatinduces cell death, or an antibody that induces apoptosis.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN™ cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2±89 and CBI-TMI);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin (1^(I) and calicheamicin 2^(I) ₁ (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; bisphosphonates, such as clodronate; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromomophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.) andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chlorambucil;GEMZAR™ gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE™ vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADE™ tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON™ toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE™megestrol acetate, AROMASIN exemestane, formestane, fadrozole, RIVISOR™vorozole, FEMARA™ letrozole, and ARIMIDEX™ anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Raf, and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN™ vaccine, LEUVECTIN™ vaccine, andVAXID™ vaccine; PROLEUKIN™ rIL-2; LURTOTECAN™ topoisomerase I inhibitor;ABARELIX™ rGnRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell in vitro and/or in vivo.Thus, the growth inhibitory agent may be one that significantly reducesthe percentage of cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), TAXOL® paclitaxel, and topo II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest GI also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (W B Saunders:Philadelphia, 1995), especially p. 13.

Examples of “growth inhibitory” anti-HER2 antibodies are those whichbind to HER2 and inhibit the growth of cancer cells overexpressing HER2.Preferred growth inhibitory anti-HER2 antibodies inhibit growth of SKBR3breast tumor cells in cell culture by greater than 20%, and preferablygreater than 50% (e.g., from about 50% to about 100%) at an antibodyconcentration of about 0.5 to 30 μg/ml, where the growth inhibition isdetermined six days after exposure of the SKBR3 cells to the antibody(see U.S. Pat. No. 5,677,171 issued Oct. 14, 1997).

An antibody which “induces cell death” is one which causes a viable cellto become nonviable. The cell is generally one which expresses theantigen to which the antibody binds, especially where the celloverexpresses the antigen. Preferably, the cell is a cancer cell, e.g.,a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney,colon, thyroid, pancreatic or bladder cell. In vitro, the cell may be aSKBR3, BT474, Calu 3, MDA-MB-453, MDA-MB-361 or SKOV3 cell. Cell deathin vitro may be determined in the absence of complement and immuneeffector cells to distinguish cell death induced by antibody dependentcell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity(CDC). Thus, the assay for cell death may be performed using heatinactivated serum (i.e. in the absence of complement) and in the absenceof immune effector cells. To determine whether the antibody is able toinduce cell death, loss of membrane integrity as evaluated by uptake ofpropidium iodide (PI), trypan blue (see Moore et al. Cytotechnology,17:1-11 (1995)) or 7AAD can be assessed relative to untreated cells.

An antibody that “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is one which expresses the antigen to which the antibody binds andmay be one which overexpresses the antigen. The cell may be a tumorcell, e.g., a breast, ovarian, stomach, endometrial, salivary gland,lung, kidney, colon, thyroid, pancreatic or bladder cell. In vitro, thecell may be a SKBR3, BT474, Calu 3 cell, MDA-MB-453, MDA-MB-361 or SKOV3cell. Various methods are available for evaluating the cellular eventsassociated with apoptosis. For example, phosphatidyl serine (PS)translocation can be measured by annexin binding; DNA fragmentation canbe evaluated through DNA laddering as disclosed in the example herein;and nuclear/chrornatin condensation along with DNA fragmentation can beevaluated by any increase in hypodiploid cells. Preferably, the antibodywhich induces apoptosis is one which results in about 2 to 50 fold,preferably about 5 to 50 fold, and most preferably about 10 to 50 fold,induction of annexin binding relative to untreated cell in an annexinbinding assay using cells expressing the antigen to which the antibodybinds.

Examples of antibodies that induce apoptosis include the anti-HER2monoclonal antibodies 7F3 (ATCC HB-12216), and 7C2 (ATCC HB 12215),including humanized and/or affinity matured variants thereof; theanti-DR5 antibodies 3F11.39.7 (ATCC HB-12456); 3H3.14.5 (ATCC HB-12534);3D5.1.10 (ATCC HB-12536); and 3H3.14.5 (ATCC HB-12534), includinghumanized and/or affinity matured variants thereof; the human anti-DR5receptor antibodies 16E2 and 20E6, including affinity matured variantsthereof (WO98/51793, expressly incorporated herein by reference); theanti-DR4 antibodies 4E7.24.3 (ATCC HB-12454); 4H6.17.8 (ATCC HB-12455);1H5.25.9 (ATCC HB-12695); 4G7.18.8 (ATCC PTA-99); and 5G11.17.1 (ATCCHB-12694), including humanized and/or affinity matured variants thereof.

In order to screen for antibodies which bind to an epitope on an antigenbound by an antibody of interest, a routine cross-blocking assay such asthat described in Antibodies, A Laboratory Manual, eds. Harlow and Lane(New York: Cold Spring Harbor Laboratory, 1988) can be performed.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to thepolypeptide. The label may be itself be detectable (e.g., radioisotopelabels or fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

B. Modes for Carrying Out the Invention

The present invention relates to various peptides having the function ofantagonizing IGF-1. Specifically, one family of such peptides (family 1)comprises the sequence:(Xaa)₁Xaa)₂Cys(Xaa)₃(Xaa)₄SerVal(Xaa)₅AlaLeu(Xaa)₆(Xaa)₇CysMet(Xaa)₈(SEQ ID NO:1) where (Xaa)₁, (Xaa)₂, and (Xaa)₇ are any amino acid,(Xaa)₃ is Phe, Leu, or Tyr, (Xaa)₄ is Glu, Asp, Ala, Gly, Thr, or Ser,(Xaa)₅ is Glu, Asp, Ala, or Gly, (Xaa)₆ is Arg or Lys, and (Xaa)₈ is Tyror Arg. (Xaa)₄ is Glu, Ala, Gly, Thr, or Ser, (Xaa)₅ is Glu, Ala, orGly, and (Xaa)₈ is Tyr. The preferred peptides of the above sequence aresuch that (Xaa)₄ is Glu, Ala or Thr, (Xaa)₅ is Ala or Gly, and Xaa8 isTyr. More preferred are the peptides wherein (Xaa)₄ is Glu or Ala,(Xaa)₅ is Ala or Gly, and (Xaa)₈ is Tyr. Still more preferred are thepeptides comprising the sequence RNCFESVAALRRCMYG (SEQ ID NO:2),MDCLASVEALKWCMYG (SEQ ID NO:3), or FECLTSVEALRGCMYG (SEQ ID NO:4). Mostpreferred are peptides that comprise SEQ ID NO:2 or 3.

The second family of such peptides (family 2) comprises the sequence:(Xaa)₁(Xaa)₂Cys(Xaa)₃(Xaa)₄Asp(Xaa)₅(Xaa)₆Gly(Xaa)₇(Xaa)₈TyrCysTrp(Xaa)₉(SEQ ID NO:5), where (Xaa)₁, (Xaa)₄, and (Xaa)₉ are any amino acid,(Xaa)₂ is Arg, Lys, Gly, Ser, or Thr, (Xaa)₃ is Ala or Val, (Xaa)₅ isAla or Leu, (Xaa)₆ is Ala, Gly, or Leu, (Xaa)₇ is Phe, Tyr, Trp, or Gly,and (Xaa)₉ is Glu, Asp, Ala, or Gly. The preferred peptides herein aresuch that (Xaa)₂ is Gly, Ser, Arg, or Thr, and (Xaa)₉ is Glu, Ala, orAsp. More preferred are peptides wherein (Xaa)₂ is Glu or Arg, (Xaa)₅ isLeu, (Xaa)₆ is Ala or Gly, (Xaa)₇ is Phe, and (Xaa)₉ is Ala. The mostpreferred of this family of peptides are those that comprise thesequence LGCASDLAGFWYCWAG (SEQ ID NO:6) or WRCVDDLGGFQYCWAG (SEQ IDNO:7).

Preferably, all the amino acids in these two families of peptides areL-amino acids. Also preferred is that these families of peptidescomprise a glycine residue after (Xaa)₈ for family 1 above or after(Xaa)₉ for family 2 above.

Production of Peptides

The peptides of this invention can be made by chemical synthesis or byemploying recombinant technology. These methods are known in the art.Chemical synthesis, especially solid-phase synthesis, is preferred forshort (e.g., less than 50 residues) peptides or those containingunnatural or unusual amino acids such as D-Tyr, ornithine, amino-adipicacid, and the like. Recombinant procedures are preferred for longerpolypeptides. When recombinant procedures are selected, a synthetic genemay be constructed de novo or a natural gene may be mutated by, forexample, cassette mutagenesis.

A useful method for identification of certain residues or regions of thepeptides herein suitable for amino acid substitution other than thosedescribed herein is called alanine-scanning mutagenesis as described byCunningham and Wells, Science, 244:1081-1085 (1989). Here a residue orgroup of target residues are identified (e.g., charged residues such asArg, Asp, His, Lys, and Glu) and replaced by a neutral ornegatively-charged amino acid to affect the interaction of the aminoacids with the surrounding aqueous environment in or outside the cell.Those domains demonstrating functional sensitivity to the substitutionthen are refined by introducing further or other variations at or forthe sites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. For example, to optimize the performanceof a mutation at a given site, Ala-scanning or random mutagenesis may beconducted at the target codon or region and the expressed compoundscreened for the optimal combination of desired activity.

Phage display of protein or peptide libraries offers another methodologyfor the selection of compounds with improved affinity, alteredspecificity, or improved stability (Smith, Curr. Opin. Biotechnol.,2:668-673 (1991)). High affinity proteins, displayed in a monovalentfashion as fusions with the M13 gene III coat protein (Clackson et al.,Trends Biotechnol. 12:173-183 (1994)), can be identified by cloning andsequencing the corresponding DNA packaged in the phagemid particlesafter a number of rounds of binding selection.

Other peptides include the fusion to the N- or C-terminus of thepeptides described herein of immunogenic polypeptides, e.g., bacterialpolypeptides such as beta-lactamase or an enzyme encoded by E. coli Trplocus or yeast protein, and C-terminal fusion with proteins having along half-life such as immunoglobulin constant region or otherimmunoglobulin regions, albumin, or ferritin as described in WO 89/02922published 6 Apr. 1989. Further, free functional groups on theside-chains of the amino acid residues can also be modified byamidation, acylation, or other substitution, which can, for example,change the solubility of the peptides without affecting their activity.Set forth below are exemplary general recombinant procedures.

From a purified IGF and its amino acid sequence, for example, an IGFantagonist that is a peptidyl mutant of an IGF may be produced usingrecombinant DNA techniques. These techniques contemplate, in simplifiedform, taking the gene, either natural or synthetic, encoding thepeptide; inserting it into an appropriate vector; inserting the vectorinto an appropriate host cell; culturing the host cell to causeexpression of the gene; and recovering or isolating the peptide producedthereby. Preferably, the recovered peptide is then purified to asuitable degree.

Somewhat more particularly, the DNA sequence encoding a peptidyl IGFantagonist is cloned and manipulated so that it may be expressed in aconvenient host. DNA encoding parent polypeptides can be obtained from agenomic library, from cDNA derived from mRNA from cells expressing thepeptide, or by synthetically constructing the DNA sequence (Sambrook etal., Molecular Cloning: A Laboratory Manual (2d ed.) (Cold Spring HarborLaboratory: N.Y., 1989)).

The parent DNA is then inserted into an appropriate plasmid or vectorthat is used to transform a host cell. In general, plasmid vectorscontaining replication and control sequences derived from speciescompatible with the host cell are used in connection with those hosts.The vector ordinarily carries a replication site, as well as sequencesencoding proteins or peptides that are capable of providing phenotypicselection in transformed cells.

For example, E. coli may be transformed using pBR322, a plasmid derivedfrom an E. coli species (Mandel et al., J. Mol. Biol. 53: 154 (1970)).Plasmid pBR322 contains genes for ampicillin and tetracyclineresistance, and thus provides easy means for selection. Other vectorsinclude different features such as different promoters, which are oftenimportant in expression. For example, plasmids pKK223-3, pDR720, andpPL-lambda represent expression vectors with the tac, trp, or P_(L)promoters that are currently available (Pharmacia Biotechnology).

One preferred vector is pB0475. This vector contains origins ofreplication for phage and E. coli that allow it to be shuttled betweensuch hosts, thereby facilitating both mutagenesis and expression(Cunningham et al., Science, 243: 1330-1336 (1989); U.S. Pat. No.5,580,723). Other preferred vectors are pR1T5 and pR1T2T (PharmaciaBiotechnology). These vectors contain appropriate promoters followed bythe Z domain of protein A, allowing genes inserted into the vectors tobe expressed as fusion proteins.

Other preferred vectors can be constructed using standard techniques bycombining the relevant traits of the vectors described above. Relevanttraits include the promoter, the ribosome binding site, the decorsin orornatin gene or gene fusion (the Z domain of protein A and decorsin orornatin and its linker), the antibiotic resistance markers, and theappropriate origins of replication.

The host cell may be prokaryotic or eukaryotic. Prokaryotes arepreferred for cloning and expressing DNA sequences to produce parentIGF-1 polypeptide, segment-substituted peptides, residue-substitutedpeptides, and peptide variants. For example, E. coli K12 strain 294(ATCC No. 31446) may be used as well as E. coli B, E. coli X1776 (ATCCNo. 31537), and E. coli c600 and c600hfl, E. coli W3110 (F-, gamma-,prototrophic/ATCC No. 27325), bacilli such as Bacillus subtilis, andother enterobacteriaceae such as Salmonella typhimurium or Serratiamarcesans, and various Pseudomonas species. The preferred prokaryote isE. coli W3110 (ATCC 27325). When expressed by prokaryotes the peptidestypically contain an N-terminal methionine or a formyl methionine andare not glycosylated. In the case of fusion proteins, the N-terminalmethionine or formyl methionine resides on the amino terminus of thefusion protein or the signal sequence of the fusion protein. Theseexamples are, of course, intended to be illustrative rather thanlimiting.

In addition to prokaryotes, eukaryotic organisms, such as yeastcultures, or cells derived from multicellular organisms may be used. Inprinciple, any such cell culture is workable. However, interest has beengreatest in vertebrate cells, and propagation of vertebrate cells inculture (tissue culture) has become a reproducible procedure. TissueCulture, Academic Press, Kruse and Patterson, editors (1973). Examplesof such useful host cell lines are VERO and HeLa cells, Chinese HamsterOvary (CHO) cell lines, W138, 293, BHK, COS-7 and MDCK cell lines.

A variation on the above procedures contemplates the use of genefusions, wherein the gene encoding the desired peptide is associated, inthe vector, with a gene encoding another protein or a fragment ofanother protein. This results in the desired peptide being produced bythe host cell as a fusion with another protein or peptide. The “other”protein or peptide is often a protein or peptide that can be secreted bythe cell, making it possible to isolate and purify the desired peptidefrom the culture medium and eliminating the necessity of destroying thehost cells that arises when the desired peptide remains inside the cell.Alternatively, the fusion protein can be expressed intracellularly. Itis useful to use fusion proteins that are highly expressed.

The use of gene fusions, though not essential, can facilitate theexpression of heterologous peptides in E. coli as well as the subsequentpurification of those gene products. Harris, in Genetic Engineering,Williamson, R., Ed. (Academic Press, London, Vol. 4, 1983), p. 127;Ljungquist et al., Eur. J. Biochem., 186: 557-561 (1989) and Ljungquistet al., Eur. J. Biochem., 186: 563-569 (1989). Protein A fusions areoften used because the binding of protein A, or more specifically the Zdomain of protein A, to IgG provides an “affinity handle” for thepurification of the fused protein. See Nilsson et al., ProteinEngineering, 1: 107-113 (1987). It has also been shown that manyheterologous proteins are degraded when expressed directly in E. coli,but are stable when expressed as fusion proteins. Marston, Biochem J.240: 1 (1986).

After expression and secretion, for example, from E. coli, the fusionprotein is cleaved to yield free peptide, which can be purified from thereaction mix. The cleavage may be accomplished using chemicals, such ascyanogen bromide, which cleaves at a methionine, or hydroxylamine, whichcleaves between an Asn and Gly residue. Using standard recombinant DNAmethodology, the nucleotide base pairs encoding these amino acids may beinserted just prior to the 5′ end of the gene encoding the desiredpeptide.

Alternatively, one can employ proteolytic cleavage of fusion protein(Carter, in Protein Purification: From Molecular Mechanisms toLarge-Scale Processes, Ladisch et al., eds. (American Chemical SocietySymposium Series No. 427, 1990), Ch 13, pages 181-193; Varadarajan etal., Proc. Natl. Acad. Sci. USA, 82: 5681-5684 (1985); Castellanos-Serraet al., FEBS Letters, 378: 171-176 (1996); Nilsson et al., J.Biotechnol., 48: 241-250 (1996)).

Proteases such as Factor Xa, thrombin, subtilisin, or trypsin, or itsmutants, and a number of others have been successfully used to cleavefusion proteins. Trypsin is preferred because peptide-Z-domain fusionsare found to be readily cleaved by this protease. Detailed proceduresfor employing trypsin as protease are found in Smith, Methods in Mol.Biol., 32: 289-196 (1994). Typically, a peptide linker that is amenableto cleavage by the protease used is inserted between the “other” protein(e.g., the Z domain of protein A) and the desired peptide. Usingrecombinant DNA methodology, the nucleotide base pairs encoding thelinker are inserted between the genes or gene fragments coding for theother proteins. Proteolytic cleavage of the partially-purified fusionprotein containing the correct linker can then be carried out on eitherthe native fusion protein, or the reduced or denatured fusion protein.

The peptide may or may not be properly folded when expressed as a fusionprotein. Also, the specific peptide linker containing the cleavage sitemay or may not be accessible to the protease. These factors determinewhether the fusion protein must be denatured and refolded, and if so,whether these procedures are employed before or after cleavage.

When denaturing and refolding are needed, typically the peptide istreated with a chaotrope, such as guanidine HCl, and is then treatedwith a redox buffer, containing, for example, reduced and oxidizeddithiothreitol or glutathione at the appropriate ratios, pH, andtemperature, such that the peptide is refolded to its native structure.

As well as by recombinant methods, peptides of the invention can beconveniently prepared using solid phase peptide synthesis (Merrifield,J. Am. Chem. Soc., 85: 2149 (1964); Houghten, Proc. Natl. Acad. Sci.USA, 82: 5132 (1985)), although other equivalent chemical synthesesknown in the art are employable. Solid-phase synthesis is initiated fromthe C-terminus of the peptide by coupling a protected ∀-amino acid to asuitable resin. Such a starting material can be prepared by attaching an∀-amino-protected amino acid by an ester linkage to a chloromethylatedresin or a hydroxymethyl resin, or by an amide bond to a BHA resin orMBHA resin. The preparation of the hydroxymethyl resin is described byBodansky et al., Chem. Ind. (London), 38: 1597-1598 (1966).Chloromethylated resins are commercially available from BioRadLaboratories, Richmond, Calif. and from Lab. Systems, Inc. Thepreparation of such a resin is described by Stewart et al., Solid PhasePeptide Synthesis (Freeman & Co., San Francisco 1969), Chapter 1, pp.1-6. BHA and MBHA resin supports are commercially available and aregenerally used only when the desired polypeptide being synthesized hasan unsubstituted amide at the C-terminus.

The amino acids are coupled to the peptide chain using techniques wellknown in the art for the formation of peptide bonds. One method involvesconverting the amino acid to a derivative that will render the carboxylgroup more susceptible to reaction with the free N-terminal amino groupof the peptide fragment. For example, the amino acid can be converted toa mixed anhydride by reaction of a protected amino acid withethylchloroformate, phenyl chloroformate, sec-butyl chloroformate,isobutyl chloroformate, pivaloyl chloride or like acid chlorides.Alternatively, the amino acid can be converted to an active ester suchas a 2,4,5-trichlorophenyl ester, a pentachlorophenyl ester, apentafluorophenyl ester, a p-nitrophenyl ester, a N-hydroxysuccinimideester, or an ester formed from 1-hydroxybenzotriazole

Another coupling method involves use of a suitable coupling agent suchas N,N′-dicyclohexylcarbodiimide or N,N′-diisopropyl-carbodiimide. Otherappropriate coupling agents, apparent to those skilled in the art, aredisclosed in E. Gross & J. Meienhofer, The Peptides: Analysis Structure,Biology, Vol. 1: Major Methods of Peptide Bond Formation (AcademicPress: New York, 1979).

It should be recognized that the ∀-amino group of each amino acidemployed in the peptide synthesis must be protected during the couplingreaction to prevent side reactions involving their active ∀-aminofunction. It should also be recognized that certain amino acids containreactive side-chain functional groups (e.g., sulfhydryl, amino,carboxyl, and hydroxyl) and that such functional groups must also beprotected with suitable protecting groups to prevent a chemical reactionfrom occurring at that site during both the initial and subsequentcoupling steps. Suitable protecting groups, known in the art, aredescribed in Gross and Meienhofer, The Peptides: Analysis. Structure,Biology, Vol. 3: “Protection of Functional Groups in Peptide Synthesis”(Academic Press: New York, 1981).

In the selection of a particular side-chain protecting group to be usedin synthesizing the peptides, the following general rules are followed.An ∀-amino protecting group (a) must render the ∀-amino function inertunder the conditions employed in the coupling reaction, (b) must bereadily removable after the coupling reaction under conditions that willnot remove side-chain protecting groups and will not alter the structureof the peptide fragment, and (c) must eliminate the possibility ofracemization upon activation immediately prior to coupling. A side-chainprotecting group (a) must render the side-chain functional group inertunder the conditions employed in the coupling reaction, (b) must bestable under the conditions employed in removing the ∀-amino protectinggroup, and (c) must be readily removable upon completion of the desiredamino acid peptide under reaction conditions that will not alter thestructure of the peptide chain.

It will be apparent to those skilled in the art that the protectinggroups known to be useful for peptide synthesis will vary in reactivitywith the agents employed for their removal. For example, certainprotecting groups such as triphenylmethyl and2-(p-biphenylyl)isopropyloxycarbonyl are very labile and can be cleavedunder mild acid conditions. Other protecting groups, such ast-butyloxycarbonyl (BOC), t-amyloxycarbonyl adamantyl-oxycarbonyl, andp-methoxybenzyloxycarbonyl, are less labile and require moderatelystrong acids, such as trifluoroacetic, hydrochloric, or borontrifluoride in acetic acid, for their removal. Still other protectinggroups, such as benzyloxycarbonyl (CBZ or Z), halobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl cycloalkyloxycarbonyl, andisopropyloxycarbonyl, are even less labile and require stronger acids,such as hydrogen fluoride, hydrogen bromide, or boron trifluoroacetatein trifluoroacetic acid, for their removal. Among the classes of usefulamino acid protecting groups are included:

(1) for an ∀-amino group, (a) aromatic urethane-type protecting groups,such as fluorenylmethyloxycarbonyl (FMOC) CBZ, and substituted CBZ, suchas, e.g., p-chlorobenzyloxycarbonyl, p-6-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, and p-methoxybenzyloxycarbonyl,o-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,2,6-dichlorobenzyloxycarbonyl, and the like; (b) aliphatic urethane-typeprotecting groups, such as BOC, t-amyloxycarbonyl, isopropyloxycarbonyl,2-(p-biphenylyl)-isopropyloxycarbonyl, allyloxycarbonyl and the like;(c) cycloalkyl urethane-type protecting groups, such ascyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl;and d) allyloxycarbonyl. The preferred ∀-amino protecting groups are BOCor FMOC.

(2) for the side chain amino group present in Lys, protection may be byany of the groups mentioned above in (1) such as BOC,p-chlorobenzyloxycarbonyl, etc.

(3) for the guanidino group of Arg, protection may be by nitro, tosyl,CBZ, adamantyloxycarbonyl, 2,2,5,7,8-pentamethylchroman-6-sulfonyl,2,3,6-trimethyl-4-methoxyphenylsulfonyl, or BOC.

(4) for the hydroxyl group of Ser, Thr, or Tyr, protection may be, forexample, by C1-C4 alkyl, such as t-butyl; benzyl (BZL); or substitutedBZL, such as p-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl,o-chlorobenzyl, and 2,6-dichlorobenzyl.

(5) for the carboxyl group of Asp or Glu, protection may be, forexample, by esterification using groups such as BZL, t-butyl,cyclohexyl, cyclopentyl, and the like.

(6) for the imidazole nitrogen of His, the tosyl moiety is suitablyemployed.

(7) for the phenolic hydroxyl group of Tyr, a protecting group such astetrahydropyranyl, tert-butyl, trityl, BZL, chlorobenzyl, 4-bromobenzyl,or 2,6-dichlorobenzyl is suitably employed. The preferred protectinggroup is 2,6-dichlorobenzyl.

(8) for the side-chain amino group of Asn or Gln, xanthyl (Xan) ispreferably employed.

(9) for Met, the amino acid is preferably left unprotected.

(10) for the thio group of Cys, p-methoxybenzyl is typically employed.

The C-terminal amino acid, e.g., Lys, is protected at the N-aminoposition by an appropriately-selected protecting group, in the case ofLys, BOC. The BOC-Lys-OH can be first coupled to the benzyhydrylamine orchloromethylated resin according to the procedure set forth in Horiki etal., Chemistry Letters, 165-168 (1978) or using isopropylcarbodiimide atabout 25EC for 2 hours with stirring. Following the coupling of theBOC-protected amino acid to the resin support, the ∀-amino protectinggroup is removed, as by using trifluoroacetic acid (TFA) in methylenechloride or TFA alone. The deprotection is carried out at a temperaturebetween about 0EC and room temperature. Other standard cleavingreagents, such as HCl in dioxane, and conditions for removal of specific∀-amino protecting groups are described in the literature.

After removal of the ∀-amino protecting group, the remaining ∀-amino andside-chain protected amino acids are coupled stepwise within the desiredorder. As an alternative to adding each amino acid separately in thesynthesis, some may be coupled to one another prior to addition to thesolid-phase synthesizer. The selection of an appropriate couplingreagent is within the skill of the art. Particularly suitable as acoupling reagent is N,N′-dicyclohexyl carbodiimide ordiisopropylcarbodiimide.

Each protected amino acid or amino acid sequence is introduced into thesolid-phase reactor in excess, and the coupling is suitably carried outin a medium of dimethylformamide (DMF) or CH₂Cl₂ or mixtures thereof. Ifincomplete coupling occurs, the coupling procedure is repeated beforeremoval of the N-amino protecting group prior to the coupling of thenext amino acid. The success of the coupling reaction at each stage ofthe synthesis may be monitored. A preferred method of monitoring thesynthesis is by the ninhydrin reaction, as described by Kaiser et al.,Anal. Biochem, 34: 595 (1970). The coupling reactions can be performedautomatically using well-known methods, for example, a BIOSEARCH 9500™peptide synthesizer.

Upon completion of the desired peptide sequence, the protected peptidemust be cleaved from the resin support, and all protecting groups mustbe removed. The cleavage reaction and removal of the protecting groupsis suitably accomplished simultaneously or stepwise. When the resinsupport is a chloro-methylated polystyrene resin, the bond anchoring thepeptide to the resin is an ester linkage formed between the freecarboxyl group of the C-terminal residue and one of the manychloromethyl groups present on the resin matrix. It will be appreciatedthat the anchoring bond can be cleaved by reagents that are known to becapable of breaking an ester linkage and of penetrating the resinmatrix.

One especially convenient method is by treatment with liquid anhydroushydrogen fluoride. This reagent not only will cleave the peptide fromthe resin but also will remove all protecting groups. Hence, use of thisreagent will directly afford the fully deprotected peptide. When thechloromethylated resin is used, hydrogen fluoride treatment results inthe formation of the free peptide acids. When the benzhydrylamine resinis used, hydrogen fluoride treatment results directly in the freepeptide amines. Reaction with hydrogen fluoride in the presence ofanisole and dimethylsulfide at 0^(E)C for one hour will simultaneouslyremove the side-chain protecting groups and release the peptide from theresin.

When it is desired to cleave the peptide without removing protectinggroups, the protected peptide-resin can undergo methanolysis to yieldthe protected peptide in which the C-terminal carboxyl group ismethylated. The methyl ester is then hydrolyzed under mild alkalineconditions to give the free C-terminal carboxyl group. The protectinggroups on the peptide chain then are removed by treatment with a strongacid, such as liquid hydrogen fluoride. A particularly useful techniquefor methanolysis is that of Moore et al., Peptides, Proc. Fifth Amer.Pept. Symp., M. Goodman and J. Meienhofer, Eds., (John Wiley, N.Y.,1977), p. 518-521, in which the protected peptide-resin is treated withmethanol and potassium cyanide in the presence of crown ether.

Another method for cleaving the protected peptide from the resin whenthe chloromethylated resin is employed is by ammonolysis or by treatmentwith hydrazine. If desired, the resulting C-terminal amide or hydrazidecan be hydrolyzed to the free C-terminal carboxyl moiety, and theprotecting groups can be removed conventionally.

It will also be recognized that the protecting group present on theN-terminal ∀-amino group may be removed preferentially either before orafter the protected peptide is cleaved from the support.

Purification of the polypeptides of the invention is typically achievedusing conventional procedures such as preparative high-pressure liquidchromatography (HPLC) (including reversed-phase HPLC) or other knownchromatographic techniques such as gel permeation, ion exchange,partition chromatography, affinity chromatography (including monoclonalantibody columns), or countercurrent distribution.

The peptides of this invention may be stabilized by polymerization. Thismay be accomplished by crosslinking monomer chains with polyfunctionalcrosslinking agents, either directly or indirectly, throughmulti-functional polymers. Ordinarily, two substantially identicalpolypeptides are crosslinked at their C- or N-termini using abifunctional crosslinking agent. The agent is used to crosslink theterminal amino and/or carboxyl groups. Generally, both terminal carboxylgroups or both terminal amino groups are crosslinked to one another,although by selection of the appropriate crosslinking agent the ∀-aminogroup of one polypeptide is crosslinked to the terminal carboxyl groupof the other polypeptide. Preferably, the polypeptides are substitutedat their C-termini with cysteine. Under conditions well known in the arta disulfide bond can be formed between the terminal cysteines, therebycrosslinking the polypeptide chains. For example, disulfide bridges areconveniently formed by metal-catalyzed oxidation of the free cysteinesor by nucleophilic substitution of a suitably modified cysteine residue.Selection of the crosslinking agent will depend upon the identities ofthe reactive side-chains of the amino acids present in the polypeptides.For example, disulfide crosslinking would not be preferred if cysteinewas present in the polypeptide at additional sites other than theC-terminus. Also within the scope hereof are peptides crosslinked withmethylene bridges.

Suitable crosslinking sites on the peptides, aside from the N-terminalamino and C-terminal carboxyl groups, include epsilon amino groups foundon lysine residues, as well as amino, imino, carboxyl, sulfhydryl andhydroxyl groups located on the side-chains of internal residues of thepeptides or residues introduced into flanking sequences. Crosslinkingthrough externally added crosslinking agents is suitably achieved, e.g.,using any of a number of reagents familiar to those skilled in the art,for example, via carbodiimide treatment of the polypeptide. Otherexamples of suitable multi-functional (ordinarily bifunctional)crosslinking agents are found in the literature.

The peptides of this invention also may be conformationally stabilizedby cyclization. The peptides ordinarily are cyclized by covalentlybonding the N- and C-terminal domains of one peptide to thecorresponding domain of another peptide of this invention so as to formcyclo-oligomers containing two or more iterated peptide sequences, eachinternal peptide having substantially the same sequence. Further,cyclized peptides (whether cyclo-oligomers or cyclo-monomers) arecrosslinked to form 1-3 cyclic structures having from 2 to 6 peptidescomprised therein. The peptides preferably are not covalently bondedthrough ∀-amino and main-chain carboxyl groups (head to tail), butrather are crosslinked through the side-chains of residues located inthe N- and C-terminal domains. The linking sites thus generally will bebetween the side-chains of the residues.

Many suitable methods per se are known for preparing mono- orpoly-cyclized peptides as contemplated herein. Lys/Asp cyclization hasbeen accomplished using Na-Boc-amino acids on solid-phase support withFmoc/9-fluorenylmethyl (OFm) side-chain protection for Lys/Asp; theprocess is completed by piperidine treatment followed by cyclization.Glu and Lys side-chains also have been crosslinked in preparing cyclicor bicyclic peptides: the peptide is synthesized by solid-phasechemistry on a p-methylbenzhydrylamine resin. The peptide is cleavedfrom the resin and deprotected. The cyclic peptide is formed usingdiphenylphosphorylazide in diluted methylformamide. For an alternativeprocedure, see Schiller et al., Peptide Protein Res., 25: 171-177(1985). See also U.S. Pat. No. 4,547,489.

Disulfide crosslinked or cyclized peptides are generated by conventionalmethods. The method of Pelton et al. (J. Med. Chem., 29: 2370-2375(1986)) is suitable, except that a greater proportion of cyclo-oligomersare produced by conducting the reaction in more concentrated solutionsthan the dilute reaction mixture described by Pelton et al., supra, forthe production of cyclo-monomers. The same chemistry is useful forsynthesis of dimers or cyclo-oligomers or cyclo-monomers. Also usefulare thiomethylene bridges. Lebl and Hruby, Tetrahedron Letters, 25:2067-2068 (1984). See also Cody et al., J. Med. Chem., 28: 583 (1985).

The desired cyclic or polymeric peptides are purified by gel filtrationfollowed by reversed-phase HPLC or other conventional procedures. Thepeptides are sterile filtered and formulated into conventionalpharmacologically acceptable vehicles.

The starting materials required for the processes described herein areknown in the literature or can be prepared using known methods and knownstarting materials.

If in the peptides being created carbon atoms bonded to fournon-identical substituents are asymmetric, then the compounds may existas diastereoisomers, enantiomers, or mixtures thereof. The synthesesdescribed above may employ racemates, enantiomers, or diastereomers asstarting materials or intermediates. Diastereomeric products resultingfrom such syntheses may be separated by chromatographic orcrystallization methods. Likewise, enantiomeric product mixtures may beseparated using the same techniques or by other methods known in theart. Each of the asymmetric carbon atoms, when present, may be in one oftwo configurations (R or S), and both are within the scope of thepresent invention.

The peptides described in this invention may be isolated as the freeacid or base or converted to salts of various inorganic and organicacids and bases. Such salts are within the scope of this invention.Examples of such salts include ammonium, metal salts like sodium,potassium, calcium, and magnesium; salts with organic bases likedicyclohexylamine, N-methyl-D-glucamine and the like; and salts withamino acids like arginine or lysine. Salts with inorganic and organicacids may be likewise prepared, for example, using hydrochloric,hydrobromic, sulfuric, phosphoric, trifluoroacetic, methanesulfonic,malic, maleic, fumaric acid, and the like. Non-toxic andphysiologically-compatible salts are particularly useful, although otherless desirable salts may have use in the processes of isolation andpurification.

A number of methods are useful for the preparation of the saltsdescribed above and are known to those skilled in the art. Examplesinclude reaction of the free acid or free base form of the peptide withone or more molar equivalents of the desired acid or base in a solventor solvent mixture in which the salt is insoluble; or in a solvent likewater after which the solvent is removed by evaporation, distillation orfreeze drying. Alternatively, the free acid or base form of the productmay be passed over an ion-exchange resin to form the desired salt or onesalt form of the product may be converted to another using the samegeneral process.

Use of Peptides

The peptides herein may be useful in diagnostic assays, e.g., fordetecting expression of IGF-1 in specific cells, tissues, or serum.

For diagnostic applications, the peptide typically will be labeled witha detectable moiety. Numerous labels are available which can begenerally grouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, I¹²⁵, ³H, and ¹³¹I. The peptide canbe labeled with the radioisotope using the techniques described inCurrent Protocols in Immunology, Volumes 1 and 2, Coligen et al., ed.(Wiley-Interscience: New York, 1991), for example, and radioactivity canbe measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the peptide using the techniquesdisclosed in Current Protocols in Immunology, supra, for example.Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate that can bemeasured using various techniques. For example, the enzyme may catalyzea color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), 73:147-166 (Academic Press, New York, 1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-nitrophenyl phosphate aschromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-p-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the peptide. Theskilled artisan will be aware of various techniques for achieving this.For example, the peptide can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the peptide in this indirect manner.Alternatively, to achieve indirect conjugation of the label with thepeptide, the peptide is conjugated with a small hapten (e.g., digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten peptide (e.g., anti-digoxin antibody). Thus,indirect conjugation of the label with the peptide can be achieved.

In another embodiment of the invention, the peptide need not be labeled,and the presence thereof can be detected using a labeled antibody thatbinds to the peptide.

The peptide of the present invention may be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: AManual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The peptide may also be used for in vivo diagnostic assays. Generally,the peptide is labeled with a radionuclide (such as ¹¹¹In, ⁹⁹Tc, ¹⁴C,¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) so that the antigen or cells expressing itcan be localized using immunoscintiography.

The peptides of this invention are shown to bind to IGF-1 and inhibitIGFBP-3 and IGFBP-1 binding to IGF-1. It is contemplated that thepeptide of the present invention may be used to treat a mammal, e.g. apatient suffering from, or predisposed to, a disease or disorder whocould benefit from administration of the peptide. It is known to thoseskilled in the art that there are many disorders caused by IGFs. Thesedisorders are set forth above.

If the disorder is cancer comprising a tumor with an IGF receptor, theefficacy of the treatment can be evidenced by a reduction in clinicalmanifestations or symptoms, including, for example, the size of a tumoror reductions in the amount of IGF available for binding to an IGFreceptor of the tumor. Examples of these protocols are well known in theart. For example, the peptide can be administered to subjects having anIGF-dependent tumor, and tumor size could be monitored using imagingtechniques, such as MRI, mammography, or ultrasound depending on thetype of tumor. Imaging could be performed, for example, twice monthly.Serum levels of IGF and IGFBP could also be measured from serum samplesfrom the subject at regular intervals. For example, levels of plasmaIGF-1 and IGF-2 in treated subjects can be monitored withradioimmunoassay, using an antibody specific for IGF and preferably forthe IGFBP binding domain on IGF. Plasma IGF levels could be measuredwith and without acid dissociation of IGFs and IGFBPs in order to assessthe levels of bound and unbound IGF. Thus, by comparing the IGF levelswith and without acid dissociation, the amount of unbound IGF can bedetermined. Normal serum levels of IGF-1 and IGF-2 after aciddissociation typically range from about 90 to 320 and 288-740:g/L,respectively. Plasma levels of the IGF antagonist peptide herein can beassessed similarly using a high affinity monoclonal antibody specificfor the IGF antagonist peptide.

The peptides of this invention may be administered to the mammal by anysuitable technique, including oral, intraventricular, transdermal,extracorporeal, parenteral (e.g., intradermal, intramuscular,intraperitoneal, intravenous, intratracheal, or subcutaneous injectionor infusion, or implant), nasal, pulmonary, vaginal, rectal, sublingual,or topical routes of administration, and can be formulated in dosageforms appropriate for each route of administration. The specific routeof administration will depend, e.g., on the medical history of thepatient, including any perceived or anticipated side effects using thepeptide, the type of peptide being administered, and the particulardisorder to be corrected. Most preferably, the administration is orallyor by pulmonary administration or continuous infusion (using, e.g.,slow-release devices or minipumps such as osmotic pumps or skinpatches), or by injection (using, e.g., intravenous or subcutaneousmeans). A specific method for administering can be found in, e.g., U.S.Pat. No. 6,124,259.

The peptide to be used in the therapy will be formulated, dosed, andadministered in a fashion consistent with good medical practice, takinginto account the particular mammal being treated, the clinical conditionof the individual patient (especially the side effects of treatment withthe peptide), the type and cause of disorder being treated, the type ofparticular peptide used, the site of delivery of the peptide, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners. The “effective amount” of the peptide tobe administered for purposes herein are thus determined by suchconsiderations, and is the minimum amount necessary to prevent,ameliorate, or treat the disorder herein, resulting in bioavailabilityof the drug to the mammal and the desired effect. As a generalproposition, the total pharmaceutically effective amount of the IGF-1antagonist peptide administered parenterally per dose will be in a rangethat can be measured by a dose-response curve.

Depending on the type and severity of the disease, about 1 μg/kg to 1000mg/kg of body weight once per day of peptide is an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. A typicaldaily dosage might range from about 1 μg/kg to 100 mg/kg or more,depending on the factors mentioned above, more preferably about 0.1 to20 mg/kg of body weight, and, when administered subcutaneously orintramuscularly, about 0.1 to 10 mg/kg of body weight. Necessarymodifications in this dosage range may be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein. See Remington's Pharmaceutical Sciences, 16th edition, Osol, ed.(Mack Publishing Co., Easton, Pa., 1980). For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.

The peptide is suitably administered by a sustained-release system.Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22, 547-556(1983)), poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed.Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12: 98-105(1982)), ethylene vinyl acetate (Langer et al., supra) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include a liposomally-entrapped peptide. Liposomescontaining the peptide are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034(1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641;Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545;and EP 102,324. Ordinarily, the liposomes are of the small (from orabout 200 to 800 Angstroms) unilamellar type in which the lipid contentis greater than about 30 mol. percent cholesterol, the selectedproportion being adjusted for the most efficacious therapy. See also themicroencapsulation technique of Langer, Nature, 392:5-10 (1998). Theactive ingredients may also be entrapped in microcapsule prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Peptides derivatized with polyethylene glycol (PEG) having a longer lifecan also be employed, based on, e.g., the conjugate technology describedin WO 95/32003 published Nov. 30, 1995.

For parenteral administration, in one embodiment, the peptide herein isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically, or parenterally, acceptable carrier, i.e., one that isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation. For example,the formulation preferably does not include oxidizing agents and othercompounds that are known to be deleterious to polypeptides.

Depending on the intended mode of administration, the peptides of thepresent invention can be in pharmaceutical compositions in the form ofsolid, semi-solid or liquid dosage forms, such as, for example, tablets,suppositories, pills, capsules, powders, liquids, suspensions, or thelike, preferably in unit dosage form suitable for single administrationof a precise dosage. The compositions will include, as noted above, aneffective amount of the selected antagonist in combination with apharmaceutically acceptable carrier and, in addition, may include othermedicinal agents, pharmaceutical agents, carriers, adjuvants, diluents,etc. By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected antagonist withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

For solid compositions, conventional nontoxic solid carriers include,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose,magnesium carbonate, and the like. Liquid pharmaceutically administrablecompositions can, for example, be prepared by dissolving, dispersing,etc. the peptide as described herein and optional pharmaceuticaladjuvants in an excipient, such as, for example, water, saline, aqueousdextrose, glycerol, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like, for example, sodium acetate, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, etc. Actualmethods of preparing such dosage forms are known, or will be apparent,to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, supra.

For oral administration, fine powders or granules may contain diluting,dispersing, and/or surface active agents, and may be presented in wateror in a syrup, in capsules or sachets in the dry state, or in anonaqueous solution or suspension wherein suspending agents may beincluded, in tablets wherein binders and lubricants may be included, orin a suspension in water or a syrup. Where desirable or necessary,flavoring, preserving, suspending, thickening, or emulsifying agents maybe included. Tablets and granules are preferred oral administrationforms, and these may be coated.

Parenteral administration, if used, is generally characterized byinjection. Injectables can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. A morerecently revised approach for parenteral administration involves use ofa slow release or sustained release system, such that a constant levelof dosage is maintained.

Generally, the formulations are prepared by contacting the peptideuniformly and intimately with liquid carriers or finely-divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, a buffered solution, and dextrose solution.Non-aqueous vehicles such as fixed oils and ethyl oleate are also usefulherein.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; glycine; amino acids such as glutamic acid,aspartic acid, histidine, or arginine; monosaccharides, disaccharides,and other carbohydrates including cellulose or its derivatives, glucose,mannose, trehalose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; counter-ions such as sodium;non-ionic surfactants such as polysorbates, poloxamers, or polyethyleneglycol (PEG); and/or neutral salts, e.g., NaCl, KCl, MgCl₂, CaCl₂, etc.

The peptide is typically formulated in such vehicles at a pH of fromabout 4.5 to 8. It will be understood that use of certain of theforegoing excipients, carriers, or stabilizers will result in theformation of salts of the peptide. The final preparation may be a stableliquid or lyophilized solid.

The peptide to be used for therapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticcompositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The peptide ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-mL vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous solution of peptide, and the resultingmixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized peptide using bacteriostaticWater-for-Injection.

A preferred route of administration of the present invention is in theaerosol or inhaled form. The peptides of the present invention, combinedwith a dispersing agent, or dispersant, can be administered in anaerosol formulation as a dry powder or in a solution or suspension witha diluent.

Suitable dispersing agents are well known in the art, and include butare not limited to surfactants and the like. For example, surfactantsthat are generally used in the art to reduce surface-induced aggregationof the peptide caused by atomization of the solution forming the liquidaerosol may be used. Non-limiting examples are surfactants such aspolyoxyethylene fatty acid esters and alcohols and polyoxyethylenesorbitan fatty acid esters. Amounts of surfactants used will vary, beinggenerally within the range of about 0.001 and 4% by weight of theformulation. In a specific aspect, the surfactant is polyoxyethylenesorbitan monooleate or sorbitan trioleate. Suitable surfactants are wellknown in the art, and can be selected on the basis of desiredproperties, depending on the specific formulation, concentration of thepeptide, diluent (in a liquid formulation), or form of powder (in a drypowder formulation), etc.

Moreover, depending on the choice of the peptide, the desiredtherapeutic effect, the quality of the lung tissue (e.g., diseased orhealthy lungs), and numerous other factors, the liquid or dryformulations can comprise additional components, as discussed furtherbelow.

The liquid aerosol formulations generally contain the peptide and adispersing agent in a physiologically-acceptable diluent. The dry powderaerosol formulations of the present invention consist of a finelydivided solid form of the peptide and a dispersing agent. With eitherthe liquid or dry powder aerosol formulation, the formulation must beaerosolized. That is, it must be broken down into liquid or solidparticles to ensure that the aerosolized dose actually reaches thealveoli. In general, the mass median dynamic diameter will be about 5micrometers or less in order to ensure that the drug particles reach thelung alveoli (Wearley, Crit. Rev. in Ther. Drug Carrier Systems, 8: 333(1991)). Aerosol particles are the liquid or solid particles suitablefor pulmonary administration, i.e., that will reach the alveoli. Otherconsiderations such as construction of the delivery device, additionalcomponents in the formulation, and particle characteristics areimportant. These aspects of pulmonary administration of a drug are wellknown in the art, and manipulation of formulations, aerosolizationmeans, and construction of a delivery device require at most routineexperimentation by one of ordinary skill in the art.

With regard to construction of the delivery device, any form ofaerosolization known in the art, including but not limited tonebulization, atomization, or pump aerosolization of a liquidformulation, and aerosolization of a dry powder formulation, can be usedin the practice of the invention. A delivery device that is uniquelydesigned for administration of solid formulations is envisioned. Often,the aerosolization of a liquid or a dry powder formulation will requirea propellent. The propellent may be any propellant generally used in theart. Specific nonlimiting examples of such useful propellants include achloroflourocarbon, a hydrofluorocarbon, a hydochlorofluorocarbon, or ahydrocarbon, including trifluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof.

In a preferred aspect of the invention, the device for aerosolization isa metered dose inhaler. A metered dose inhaler provides a specificdosage when administered, rather than a variable dose depending onadministration. Such a metered dose inhaler can be used with either aliquid or a dry powder aerosol formulation. Metered dose inhalers arewell known in the art.

Once the peptide reaches the lung, a number of formulation-dependentfactors affect the drug absorption. It will be appreciated that intreating a disease or disorder that requires circulatory levels of thepeptide, such factors as aerosol particle size, aerosol particle shape,the presence or absence of infection, lung disease, or emboli may affectthe absorption of the peptides. For each of the formulations describedherein, certain lubricators, absorption enhancers, protein stabilizersor suspending agents may be appropriate. The choice of these additionalagents will vary depending on the goal. It will be appreciated that ininstances where local delivery of the peptides is desired or sought,such variables as absorption enhancement will be less critical.

The liquid aerosol formulations of the present invention will typicallybe used with a nebulizer. The nebulizer can be either compressed airdriven or ultrasonic. Any nebulizer known in the art can be used inconjunction with the present invention such as but not limited to the:ULTRAVENT™ nebulizer (Mallinckrodt, Inc., St. Louis, Mo.) or the ACORNII™ nebulizer (Marquest Medical Products, Englewood Colo.). Othernebulizers useful in conjunction with the present invention aredescribed in U.S. Pat. Nos. 4,624,251; 3,703,173; 3,561,444; and4,635,627.

The liquid aerosol formulation may include a carrier. The carrier is amacromolecule that is soluble in the circulatory system and that isphysiologically acceptable where physiological acceptance means thatthose of skill in the art would accept injection of said carrier into apatient as part of a therapeutic regime. The carrier preferably isrelatively stable in the circulatory system with an acceptable plasmahalf life for clearance. Such macromolecules include but are not limitedto soya lecithin, oleic acid, and sorbitan trioleate, with sorbitantrioleate preferred.

The liquid aerosol formulations herein may also include other agentsuseful for protein stabilization or for the regulation of osmoticpressure. Examples of the agents include but are not limited to salts,such as sodium chloride or potassium chloride, and carbohydrates, suchas glucose, galactose or mannose, and the like.

It is also contemplated that the present pharmaceutical formulation willbe used as a dry powder inhaler formulation comprising a finely dividedpowder form of the peptide and a dispersant. The form of the peptidewill generally be a lyophilized powder. Lyophilized forms of peptidescan be obtained through standard techniques.

In another embodiment, the dry powder formulation will comprise a finelydivided dry powder containing one or more peptides of the presentinvention, a dispersing agent and also a bulking agent. Bulking agentsuseful in conjunction with the present formulation include such agentsas lactose, sorbitol, sucrose, or mannitol, in amounts that facilitatethe dispersal of the powder from the device.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Hence, the present application contemplates combining the peptide withone or more other therapeutic agent(s), which depend on the particularindication being treated. While the agent may be an endocrine agent suchas a GH, a GHRP, a GHRH, a GH secretagogue, an IGFBP, ALS, a GHcomplexed with a GHBP, it is preferably a cytotoxic agent, especiallyfor treating cancer. For instance, the peptide may be co-administeredwith another peptide (or multivalent antibodies), a monovalent orbivalent antibody (or antibodies), chemotherapeutic agent(s) (includingcocktails of chemotherapeutic agents), other cytotoxic agent(s),anti-angiogenic agent(s), cytokines, and/or growth inhibitory agent(s).Where the peptide induces apoptosis, it may be particularly desirable tocombine the peptide with one or more other therapeutic agent(s) thatalso induce apoptosis. For instance, the peptide may be combined withpro-apoptotic antibodies (e.g., bivalent or multivalent antibodies)directed against B-cell surface antigens (e.g., RITUXAN®, ZEVALIN® orBEXXAR® anti-CD20 antibodies) and/or with (1) pro-apoptotic antibodies(e.g., bivalent or multivalent antibodies directed against a receptor inthe TNF receptor superfamily, such as anti-DR4 or anti-DR5 antibodies)or (2) cytokines in the TNF family of cytokines (e.g., Apo2L). Likewise,the peptide may be administered along with anti-ErbB antibodies (e.g.,HERCEPTIN® anti-HER2 antibody) alone or combined with (1) and/or (2).Alternatively, or additionally, the patient may receive combinedradiation therapy (e.g., external beam irradiation or therapy with aradioactive labeled agent, such as an antibody), ovarian ablation,chemical or surgical, or high-dose chemotherapy along with bone marrowtransplantation or peripheral-blood stem-cell rescue or transplantation.Such combined therapies noted above include combined administration(where the two or more agents are included in the same or separateformulations), and separate administration, in which case,administration of the peptide can occur prior to, and/or following,administration of the adjunct therapy or therapies. The effective amountof such other agents depends on the amount of peptide present in theformulation, the type of disorder or treatment, and other factorsdiscussed above. These are generally used in the same dosages and withadministration routes as used hereinbefore or about from 1 to 99% of theheretofore employed dosages.

Optionally, before the peptide is administered to the mammal, the levelsof various markers are measured to determine disease state. Hence,assays can be used for measuring IGF levels, particularly IGF-1 levelsas a measure of predicting, diagnosing, and monitoring a disorder suchas cancer. A strong consistent positive association between IGF-1 andbreast or prostate cancer risk has been observed, especially withadjustment for IGFBP-3. See WO 99/38011. High levels of IGF-1 arepredictive of increased risk for prostate cancers, whereas IGFBP has aprotective effect. Additionally, the IGF or IGF/IGFBP assay can becombined with a test for prostate-specific antigen (PSA) for improvedability to predict patient prognosis and monitor treatment. The methodinvolves measuring the concentration of IGF or IGFBP-3 and/or PSA in abody sample from a mammal, wherein changes in the concentration of suchcomponents as compared to normal reference values indicate an increasedrisk for prostate cancer.

In one embodiment, before treatment the concentration of IGF-1 ismeasured in a body sample from the mammal, wherein an elevatedconcentration of IGF-1 above a reference range for IGF-1 indicates anincreased risk for prostate cancer.

In another embodiment, the method involves measuring the concentrationof IGF-1 and IGFBP in a specimen from an individual, wherein increasedIGF-1 and decreased IGFBP, as compared to a normal reference rangevalue, indicates an increased risk for prostate cancer.

In yet another embodiment, the method involves measuring the IGF/PSAstatus of an individual. High IGF and PSA levels and/or low IGFBP levelsare indicative of individuals at risk for severe prostate cancer or whohave prostate cancer with a poor prognosis.

A multivariate adjustment of the IGF-1 concentration relative to theIGFBP-3 concentration provides an adjusted IGF-1 level that can becompared to an adjusted normal reference range value. An algorithm canbe designed, by those skilled in the art of statistical analyses, whichwill allow the user to quickly calculate an adjusted IGF level or IGFstatus for use in making predictions or monitoring prostate disease.With additional patient data, generated similarly to the mannerdescribed herein, it will be possible to more accurately define normalreference range values for IGF status parameters. The algorithm andnormal reference values can be used to generate a device that will allowthe end user to input IGF, IGFBP, and quickly and easily determine theIGF status or risk index of an individual. Similarly, it is possible toprovide a device that indicates the IGF/PSA status of an individual.

The IGF status is reflected in the levels of IGF and IGFBP. For example,a high IGF status is reflected by high levels of IGF and stimulators ofIGF activity and low levels of inhibitors of IGF activity such as IGFBP.The IGF status of an individual is now known to vary—either up ordown—in certain conditions involving the prostate, including but notlimited to prostate adenocarcinoma and benign prostatic hyperplasia. TheIGF/PSA status is a combination of IGF status and PSA levels.Individuals with high IGF/PSA status are at risk for developing severeprostate cancer. High IGF and PSA levels and low IGFBP levels reflect ahigh IGF/PSA status.

“Prostate disease” includes diseases or disorders associated withpathologic conditions of the prostate, including but not limited to,prostate cancer or benign prostatic hyperplasia. The method here ispreferably used to determine the risk of an individual developingprostate cancer.

The body sample collected from the mammal may be taken by any method,including venipuncture or capillary puncture, or biopsy, and thespecimen collected into an appropriate container for receiving thespecimen. Alternative, the specimen may be placed onto filter paper.

The IGF and IGFBP and/or PSA can be measured by techniques well known tothose skilled in the art, including immunoassays such as enzyme-linkedimmunosorbent assay (ELISA), enzyme immunoassay (EIA), fluorescencepolarization immunoassay (FPIA), fluorescence immunoassay (FIA), andradioimmunoassay (RIA). The assays described in, for example, U.S. Pat.Nos. 5,935,775; 6,066,464; and 5,747,273; Zapp et al., J. Clin. Invest.,68: 1321-1330 (1981); and EP 700,994) are particularly suitable herein.Further, the concentrations of the IGF, IGFBP, and/or PSA may, forexample, be measured by test kits supplied by Diagnostic SystemsLaboratories, Inc., Webster, Tex. In a preferred embodiment, total IGF-1can be measured. In some cases, it may be advantageous to measure total,bound, and/or free IGF-1. For example, suitable highly specific andsimple non-competitive ELISAs for reliable determination of IGF-1(Khosravi et al., Clin. Chem., 42: 1147-54 (1996)), IGFBP-3 (Khosravi etal., Clin. Chem., S6:234 (1996)), and IGFBP-1 (Khosravi et al., Clin.Chem., S6:171 (1996)) have been described. The high-affinity antibodiesincorporated in these immunoassays have been selected for lack ofcross-reactivity or interference by the closely related peptides orbinding protein.

Men in the highest quartile of circulating IGF-1 have a relative risk ofprostate cancer of 4.32 (95 percent confidence interval 1.76-10.6)compared to men in the lowest quartile. There was a significant lineartrend such that a 100 ng/ml increase in IGF-1 level was associated witha doubling of risk (p=0.001). Furthermore, this association is evidentamong men with normal as well as elevated baseline PSA levels. Theseresults indicate that circulating IGF-1 is a predictor of prostatecancer risk.

In addition, the invention contemplates using gene therapy for treatinga mammal, using nucleic acid encoding the IGF antagonist peptide.Generally, gene therapy is used to increase (or overexpress) IGF levelsin the mammal. Nucleic acids that encode the IGF antagonist peptide canbe used for this purpose. Once the amino acid sequence is known, one cangenerate several nucleic acid molecules using the degeneracy of thegenetic code, and select which to use for gene therapy.

There are two major approaches to getting the nucleic acid (optionallycontained in a vector) into the patient's cells for purposes of genetherapy: in vivo and ex vivo. For in vivo delivery, the nucleic acid isinjected directly into the patient, usually at the site where the IGFantagonist peptide is required. For ex vivo treatment, the patient'scells are removed, the nucleic acid is introduced into these isolatedcells and the modified cells are administered to the patient eitherdirectly or, for example, encapsulated within porous membranes which areimplanted into the patient. See, e.g., U.S. Pat. Nos. 4,892,538 and5,283,187. There are a variety of techniques available for introducingnucleic acids into viable cells. The techniques vary depending uponwhether the nucleic acid is transferred into cultured cells in vitro orin vivo in the cells of the intended host. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjection, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, etc. Acommonly used vector for ex vivo delivery of the gene is a retrovirus.

The currently preferred in vivo nucleic acid transfer techniques includetransfection with viral vectors (such as adenovirus, Herpes simplex Ivirus, or adeno-associated virus) and lipid-based systems (useful lipidsfor lipid-mediated transfer of the gene are DOTMA, DOPE, and DC-Chol,for example). In some situations it is desirable to provide the nucleicacid source with an agent that targets the target cells, such as anantibody specific for a cell surface membrane protein or the targetcell, a ligand for a receptor on the target cell, etc. Where liposomesare employed, proteins that bind to a cell-surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake. Such proteins include, e.g., capsid proteins orfragments thereof tropic for a particular cell type, antibodies forproteins that undergo internalization in cycling, and proteins thattarget intracellular localization and enhance intracellular half-life.The technique of receptor-mediated endocytosis is described, forexample, by Wu et al., J. Biol. Chem., 262: 4429-4432 (1987) and Wagneret al., Proc. Natl. Acad. Sci. USA 87: 3410-3414 (1990). For review ofthe currently known gene marking and gene therapy protocols, seeAnderson et al., Science, 256: 808-813 (1992). See also WO 93/25673 andthe references cited therein.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture or kitcontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container andinstructions, such as a label or package or product insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, etc., preferably a vial. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition with at least the peptide herein and mayhave a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The instructions direct the user how toutilize the composition for treating the condition of choice, such ascancer. The kit may optionally include a second container with acomposition comprising a further active agent as set forth above, suchas a cytotoxic agent. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer'ssolution, and dextrose solution. It may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, and syringes.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. The disclosures of all literature and patentcitations mentioned herein are expressly incorporated by reference.

EXAMPLES

Data on a model compound (an IGF-1 mutant with amino acid changes atresidues 24 and 31 (Y24L, Y31A), also designated (Leu²⁴,Ala³¹)hIGF-1 orIGF-M) for predicting behavior of the peptides herein in vitro and invivo is also disclosed in WO 98/45427, supra. WO 98/45427 also discloseshow to dose an IGF antagonist for use in humans. From the doses of IGF-1used and the concentrations of IGFBP, IGF-1 and IGF-2 demonstrated, itis simple to calculate how much of an IGF antagonist should be given todecrease levels of active endogenous IGF. The molecular size relative toIGF-1, the affinity of the IGF antagonist for the IGF-1, and itsbioavailability would be other variables taken into account to arrive atdoses that decreased active IGF in a human.

Example 1

Experimental Procedure

Construction of Polyvalent Naïve Peptide Libraries

Libraries were constructed using the method of Sidhu et al., MethodsEnzymol., 328: 333-363 (2000) with a phagemid containing anIPTG-inducible Ptac promoter driving the expression of open readingframes encoding fusion proteins of the following form: the STIIsecretion signal (MKKNIAFLLASMFVFSIATNAYA; SEQ ID NO:8), followed by arandom peptide (i.e., a member of the naïve peptide library), followedby a linker (GGGSGGG; SEQ ID NO:9), followed by the M13 gene-8 majorcoat protein (AEGDDPAKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSK AS; SEQID NO:10).Twenty-two different peptide libraries were constructed as shown inTable 1. Phage displaying the naïve libraries were purified byprecipitation with PEG/NaCl as described in Sidhu et al., supra, andstored frozen at −70° C.Isolation of IGF-1 Binding Peptides from Naïve Peptide-Phage Libraries

IGF-1 was obtained in house as described in U.S. Pat. No. 5,342,763.

Immunosorbant plates (Nunc Maxisorp) were coated with 5 μg/ml of IGF-1in 50 mM sodium carbonate buffer (pH 9.6) for one hour at roomtemperature, followed by blocking for 1 hr with 0.2% BSA inphosphate-buffered saline (PBS). The plates were washed four times withPBS, 0.05% TWEEN®-20 detergent.

Phage from 26 naïve peptide-P8 libraries (Table 1) were pooled. Toselect peptide-phage that bound specifically to IGF-1, the library poolwas added to the above-described IGF-1-coated plate. In the first roundof selection, 4.8 mL of phage solution (about 10¹³ phage/mL) was addedto 48 coated wells (100 μl/well). After two hours incubation withshaking, the plate was washed 12 times with PBS, 0.05% TWEEN®-20detergent to remove unbound phage. Bound phage were then eluted with0.2M glycine, pH 2.0 for 5 minutes (100 μl/well), and the phage eluantwas neutralized by adding ⅙ volume of 1.0M Tris, pH8.0. The eluted phagewere amplified by propagation in E. coli XL1-blue cells with M13-VCShelper phage (Stratagene), and the amplified phage pool was cycledthrough additional rounds of binding selection. In total, four rounds ofbinding selection were performed. The procedure for round 3 wasidentical to that for round 1, while rounds 2 and 4 differed only in theuse of 0.2% Casein in place of BSA in both the blocking solution and thephage cocktail.

From each round, individual peptide-displaying phage were isolated andanalyzed for binding to IGF-1 in a phage ELISA by capturing thepeptide-phage with IGF-1 immobilized on a plate, and detecting boundphage (see below). As a control for non-specific binding, the phage werealso analyzed in a phage ELISA that used plates coated with BSA. Phagethat exhibited strong signals in the phage ELISA with IGF-1 immobilizedon plates, but not with the control ELISA, were subjected to DNAsequence analysis. The results from sequencing of positive phage clonesare seen in Table 2a.

Second-Generation Affinity Maturation of IGF-1 Binding Peptides

The IGF-1 binding peptides from the CX9C class shown in Table 2a couldbe grouped into two distinct families based on sequence homology. Basedon the sequence conservation in the two families, four second-generationlibraries were designed in which highly conserved residues were heldconstant, moderately conserved residues were represented by degeneratecodons that provided partial randomization, and unconserved residueswere represented by a codon (NNK) that encoded all twenty natural aminoacids (Table 2b). The peptide phage libraries were displayed on theN-terminus of human growth hormone (hGH) fused to P3 of the phage coatto ensure monovalency (Table 2b).

Phage from the libraries were pooled and cycled through four rounds ofbinding selections as described above. Rounds 1 and 3 used immobilizedIGF-1 as the capture target to select phage that bound specifically toIGF-1; 0.2% BSA was used in the blocking buffer and the phage cocktail.Rounds 2 and 4 used anti-hGH monoclonal antibody 3F6.B1.4B1 (Jin et al.,J. Mol. Biol., 226: 851-865 (1992)) as the capture target to select forphage that still displayed hGH and thus select against clones in whichthe hGH gene had been deleted; 0.2% Casein was used in the blockingbuffer and the phage cocktail.

Finally, a fifth round of selection was conducted in which the harvestedphage pool was incubated with 1.0 nM biotinylated IGF-1 (bio-IGF) in PBSwith 0.2% BSA for 2 hours. The phage and bio-IGF solution was added tostreptavidin-linked magnetic beads, previously blocked with BSA andwashed as above. After half an hour, the magnetic beads were washed 12times with PBS, 0.05% TWEEN®-20 detergent to remove unbound phage. Theremaining phage was eluted with 1.0 M HCl and neutralized by adding ⅙volume of 1.0 M Tris buffer, pH 8.0.

Individual phage clones from round 5 were isolated and analyzed in phageELISAs. Specifically binding phage clones were identified as those whichbound to both IGF-1 and anti-hGH but not to BSA. These positive cloneswere subjected to DNA sequence analysis and the results are shown inTable 3.

Phage ELISA

E. coli XL1-Blue harboring phagemids were grown overnight at 37° C. in2YT, 50 μg/mL carbenicillin, 10 μg/mL tetracycline and M13-VCS helperphage (10¹⁰ phage/mL). Phage were harvested from the culture supernatantby precipitation twice with PEG/NaCl and resuspended inphosphate-buffered saline, 0.2% BSA, 0.1% TWEEN®-20 detergent (BSAblocking buffer). Phage concentrations were determinedspectrophotometrically (λ₂₆₈=1.2×10⁸ M⁻¹ cm⁻¹).

MAXISORP™ immunoplates (96-well) were coated with capture target proteinfor 2 hours at room temperature (100 μL at 5 μg/mL in 50 mM carbonatebuffer, pH 9.6). The plates were then blocked for 1 h with 0.2% BSA inphosphate-buffered saline (PBS) and washed eight times with PBS, 0.05%TWEEN®-20 detergent. Phage particles were serially diluted into BSAblocking buffer and 100 μL were transferred to coated wells. After 1 h,plates were washed eight times with PBS, 0.05% TWEEN®-20 detergent,incubated with 100 μL of 1:3000 horse radish peroxidase/anti-M13antibody conjugate in BSA blocking buffer for 30 min, and then washedeight times with PBS, 0.05% TWEEN®-20 detergent and two times with PBS.Plates were developed using a tetramethylbenzidine substrate (TMB,Kirkegaard and Perry, Gaithersburg, Md.), stopped with 1.0 M H₃PO₄, andread spectrophotometrically at 45° nm.

Affinity Measurement by Monovalent Phage ELISA

A modified phage ELISA was used to estimate the binding affinities ofselected second-generation IGF-1 binding peptides displayed in amonovalent format as described in Sidhu et al., supra, and in Clacksonet al., supra. Phage ELISAs were carried out as described above, usingplates coated with IGF-1. Peptide-displaying phage were serially dilutedand binding was measured to determine a phage concentration giving <50%of the ELISA signal at saturation.

A fixed, subsaturating concentration of peptide-phage was mixed withserial dilutions of IGF-1 and incubated for 1.0 hr and then transferredto assay plates coated with IGF-1. After 30-min incubation, the plateswere washed and developed as described above. The binding affinities ofthe peptides for IGF-1 were determined as IC50 values where the IC50value is defined as the concentration of IGF-1 that blocked 50% of thepeptide-phage binding to the immobilized IGF-1. The results are shown inTable 4.

Peptide Synthesis

Peptides were synthesized by either manual or automated (Milligen 9050)solid-phase synthesis at 0.2 mM on PEG-polystyrene resin (Bodansky andBodansky, in The Practice of Peptide Synthesis (Springer-Verlag, NewYork, 1984)) utilizing Fmoc chemistry. Purification was as described inDubaquie and Lowman, Biochemistry, 38: 6386-6396 (1999)) Synthesizedpeptides are shown in Table 5.

Peptide Inhibition of Phage IGF-1 Binding to IGF Binding Proteins

For inhibition of IGFBP-1 and IGFBP3, E. coli cells (XL1-Blue,Stratagene) freshly transformed with the phage vector pIGF-g3 displayinghuman IGF-1 as described in Dubaquie and Lowman, supra, was grownovernight in 5 ml of 2YT medium (Sambrook et al., Molecular Cloning. ALaboratory Handbook (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989)). The IGF-1 displaying phage was titered against IGFbp1 and bp3 for a 500-1000× dilution for preincubation with serialdilutions the synthesized peptides and binding protein standards for 45minutes. Immunosorbant plates were coated with IGF binding proteins andblocked with 0.5% TWEEN®-20 detergent and PBS and washed as above. Thesamples were added to the plates for 20 minutes, washed and detected asabove. The experimental IC50 values are in Table 5.

Cell-Based Insulin KIRA Assay of IGF-1 Binding Peptide Activity

A kinase receptor activation assay (KIRA) (Sadick et al., J. Pharm.Biomed. Anal., 19, 883-891 (1999)) for measuring phosphorylation ofhuman insulin receptor (hIR) was developed using Chinese hamster ovarycells transfected with the hIR. (TRY-1R 5.3) Cells were grown overnightin 96-well plates with medium (PS/20) at 37° C. Supernatants weredecanted and stimulation media (PS/20 and 0.5% BSA) containing eitherpeptide samples (peptides incubated with 25 nm IGF-1 for 1 hr),experimental controls (IGF Bp1 incubated with IGF-1 for 1 hr), and 25 nmrhIGF-1 standards were added. After fifteen minute stimulation at 37°C., stimulation solutions were removed and cells were lysed with abuffer containing 50 mM HEPES, 150 mM NaCl, 0.5% Triton-X-100™octylphenyl ethylene oxide condensate, 1 mM AEBSF, aprotinin and 0.05 mMleupeptin, and 2 mM sodium orthovanadate. Lysates were frozen at −70° C.for ELISA. Immunosorbant plates were coated with 2 μg/ml insulinreceptor Ab-2 (clone 83-7) in PBS at pH 7.0 overnight at 4° C. The platewas blocked and washed as above. Cell lysates containing transfected hIRwere incubated on the capture ELISA plates for 2 hrs. After removingunbound receptor by washing, biotinylated anti-phosphotyrosine 4G10(Upstate Biologicals Inc.) was added to detect activated receptor. After2 hrs, the plates were developed with streptavidin conjugated to HRP andTMB substrate as above. The results are shown in Table 5.

Displacement of IGF-1 on MCF-7 Cells

MCF7 (ATCC-HTB, Bethesda, Md.), a breast carcinoma cell line thatexpresses IGF receptors as well as insulin receptors (Grupe et al., J.Biol. Chem., 270, 22085-22088 (1995)) was used to detect inhibition of¹²⁵I IGF-1 to receptors on cells by synthesized peptides. Cell werepassaged weekly in media containing a 50/50 mix of high-glucoseDMEM/Ham's F12, with 10% fetal bovine serum and 10 mM HEPES pH 7.2.Cells were plated and grown overnight maintained at 37° C. and 5% CO₂.For IGF-1 iodination, 50 μg of IGF-1 was diluted into 200 μL of PBS andadded to a tube coated with 100 μg of IODOGEN®1,3,4,6-tetrachloro-3α-6β-diphenylglycouril) (Pierce Chemical Co.),incubated with 1 mCi of ¹²⁵I-NaI (10 μL) at room temperature for 10-15minutes. Synthesized peptides and binding protein controls werepreincubated with 2 nm ¹²⁵I IGF-1 for 40 minutes at 37° C. The sampleswere added to cells for 30 minutes. The cells were washed with media andlysed with 1N NaOH. Fifteen-minute counts were taken and the results areshown in Table 5.

Cell-Based IGF-1 KIRA Assay

A KIRA for measuring the activation of the human type 1 IGF-1 receptorwas performed using human MCF-7 cells (as described above). Cells weregrown overnight in 96-well plates with medium (50:50 F12/DMEM, Gibco).Supernatants were decanted, and stimulation media (50:50 F12/DMEM with25 mM HEPES and 2.0% BSA) containing either controls (2 nM IGF-1preincubated with IGF Bp1 or Bp3) or experimental samples (Peptidespreincubated for 30 min. with 2 nm IGF-1) were added. After 15-minutestimulation the cells were lysed, and added to the polyclonal anti-IGF-R(3B7 Santa Cruz Biotech) coated overnight on immunosorbant plates. Thedetection ELISA was performed as above. The results are seen in Table 5.

Results: TABLE 1 Naïve phage library design and diversity (where X isany amino acid and any number following X is a multiplier) SEQ DiversityLibrary Design (P8 display) ID NO: (×10¹⁰) 1 X8 11 2.6 2 X20 12 1.2 3C—X6—C—X6—CC—X3—C—X6—C 13 1.6 4 CC—X3—C—X6—C 14 1.7 5 CC—X5—C—X4—C—X4—CC15 1.6 6 C—X—C—X7—C—X3—C—X6 16 1.5 7 X4—C—X2—GP—X4—C—X4 17 2.0 8C—X2—GP—X4—C 18 2.5 9 X7—C—X4—C—X7 19 2.5 10 X7—C—X5—C—X6 20 1.4 11X6—C—X6—C—X6 21 2.5 12 X6—C—X7—C—X5 22 2.1 13 X5—C—X8—C—X5 23 1.9 14X5—C—X9—C—X4 24 2.0 15 X4—C—X10—C—X4 25 2.5 16 X2—C—X4—C—X2 26 2.1 17X2—C—X5—C—X2 27 2.2 18 X2—C—X6—C—X2 28 1.5 19 X2—C—X7—C—X2 29 2.1 20X2—C—X8—C—X2 30 2.1 21 X2—C—X9—C—X2 31 2.2 22 X2—C—X10—C—X2 32 2.4

TABLE 2a Naive phage peptide sequences SEQ ID SCAFFOLD NO: X20 A S Q T PW P Y S I L F G E W W N A G F 33 E A G A E S R G W L Q A R C G E L L G V34 H W D W T G G Y W W I G R E P W K E A A 35 R L N A E X L R M G W G YM V W H W L S 36 CX6C G A Q A W L C E Q R E E W C G Q M L G T 37 Y D W VE A C Q K W P V L C M D S T M Y 38 CX7C G I R E E L C D K G L H K M C FR E V R 39 C E C G K V S S R G C E K L C W L V S Y M 40 CX8C D A M D C VV G P E W R K C F L E G 41 S G T A C R W G P S W L L C S L A G S P 42 GE G P E C D L R Q W G N L C G H W E T 43 L S S E E C W E A L K W Q G C LM S E R 44 S F C E F N D W W P T C L V 45 CX9C G V E T C Y S D A M N T QY C W T T E L 46 E V A R C V V D A G G T W Y C W A E M A 47 G E S T C VT D L E R V E Y C W D E K S 48 H P D K C F A D V R A L Q E C M E S V R49 R E V K C M K D L S G H E Y C W A E P R 50 S T Y S C I R D M G W A VY C W E T T L 51 V E E K C Y E S I T A L R H C M Q A M Q 52 V E S E C LL S L P N L R R C M M D R L 53 V K D E C L M S V E A L K N C M G L V S54 V M D Q C F E S Y A E M R K C M L D G S 55 I D C L D S V E A L K Q CM Y 56 I E C W Q D L Q G T R L C W E 57 G A S T T C L E K Y R E R Q W CK E L T 58 G E A A E C A Y D S L G M A Y C Y A K E 59 Q I P A G C Y E SV Q S L L E C V Q S A 60 T A G I E C A Y D K H L D Q Y C W W K E 61

TABLE 2b Second generation library construction for family I and II ofthe IGF-1 binding peptides. Library DNA Code Diversity Possible Aminoacids IGF-PEP8-FI NNK VVKPHQRTNKSADEG TGCC YWCFYLH GASDE AGCS GTCV 2.0 ×10¹⁰ All 20 IGF-PEP9-FII NNK RVKTNKSRADEG TGCC RYKIMVTA NNK GACD KYGLSVA2.6 × 10¹⁰ All 20 All 20 IGF-PEP10-FI NNK GASDE TGCC YWCFYLHRNKIMTNKSRVADEG AGCS GTCV 2.2 × 10¹⁰ All 20 IGF-PEP12-FII NNK ARGKR TGCCRYKIMVTA NNK GACD KYGLSVA 2.0 × 10¹⁰ All 20 All 20 Library DNA CodeDiversity Possible Amino acids IGF-PEP8-FI VVKPHQRTNKSADEG GCTA CTCLARGKR VVKPHQRTNKSADEG TGTC ATGM NNK 2.0 × 10¹⁰ All 20 IGF-PEP9-FII NNKGGTG NNK NNK TACY TGTC TGGW GMKADE 2.6 × 10¹⁰ All 20 All 20 All 20IGF-PEP10-FI GAGE GCTA CTCL ARGKR NNK TGTC ATGM NNK 2.2 × 10¹⁰ All 20All 20 IGF-PEP12-FII RGTGS GGTG NNK NNK TACY TGTC TGGW GMKADE 2.0 × 10¹⁰All 20 All 20

TABLE 3 Sequences from second generation library sorting SEQ ID Family INO: R N C F E S V A A L R R C M Y 62 F G C Y E S V A A L R T C M Y 63 YH C F E S V D A L R R C M K 64 L E C F K S V E A L K T C M A 65 R D C FD S V E A L R X C M Y 66 L D C F T S V E A L R W C M R 67 A E C F G S VE A L K G C M H 68 R D C F V S V E A L R H C M Y 69 H D C F A S V E A LR R C M Y 70 S D C F G S V E A L K M C M Y 71 S D C F E S V E A L R A CM Y 72 M E C H G S V E A L K I C M X 73 D E C L T S V E A L R Y C M A 74G D C L G S V E A L K M C M D 75 N D C L D S V E A L R F C M S 76 A D CL D S V E A L R R C M R 77 F E C L T S V E A L R G C M Y 78 R D C L A SV E A L R S C M Y 79 M D C L A S V E A L K W C M Y 80 L E C Y T S V E AL K W C M R 81 M D C Y S S V E A L R Y C M R 82 L T C L D S V G A L R RC M R 83 H P C L E S V G A L K A C M Y 84 N S C L E S V H A L R E C M L85 A G C L D S V K A L K R C M I 86 Y T C F E S V P A L R P C M R 87 Y TC F E S V P A L R P C M R 88 S H C F D S V R A L R H C M R 89 T S C F ES V R A L R A C M R 90 N A C L E S V R A L K A C M S 91 L T C L D S V RA L K E F M L 92 S K C L D S V S A L R R C M Q 93 R G C Y E S V T A L RH C M Y 94

Family II W R C A Q D A G G W T Y C W A 95 F R C A G D A G G R S Y C W D96 V R C A Y D A G G S R Y C W E 97 A R C A R D A G G F Y Y C W A 98 I RC V Q D A G G V R Y C W D 99 V R C V A D A G G F L Y C W A 100 W R C V TD A G G R P Y C W A 101 A S C V A D A G G G G Y C W D 102 V D C V W D AH G W G Y C W A 103 V T C A A D A L G F L Y C W E 104 L R C T E D A S GR V Y C W D 105 G G C A S D L A G F R Y C W E 106 L G C A S D L A G F WY C W A 107 Y R C A T D L A G F S Y C W A 108 K G C V S D L F G A G Y CW D 109 V R C A W D L G G R A Y C W A 110 L R C A E D L G G Y F Y C W A111 W R C V D D L G G F Q Y C W A 112 V K C A R D L S G F V Y C W A 113G G C T G D S A G P G Y C W E 114 R R C V S D S G G R T Y C W A 115 L KC A L D T F G G L Y C W A 116 R K C A S D V G G V T Y C W D 117 M S C AR D V R G V R Y C W A 118 G A C M T D V R G R E Y C W D 119 F R C A W EL G W L Y V L G L 120

TABLE 4 Positive second generation clones with phage competition IC50.SEQ ID NO: IC50 error F1-A H P C L E S V G A L K A C M Y 84 4.01 × 10⁻⁷F1-B F G C Y E S V A A L R T C M Y 63 3.01 × 10⁻⁷ F1-C R N C F E S V A AL R R C M Y 62 1.08 × 10⁻⁷ 2.63 × 10⁻⁸ F1-D H D C F A S V E A L R R C MY 70 4.68 × 10⁻⁷ 1.27 × 10⁻⁷ F1-E S D C F G S V E A L K M C M Y 71 7.11× 10⁻⁷ 4.30 × 10⁻⁷ F1-F F E C L T S V E A L R G C M Y 78 2.86 × 10⁻⁷8.37 × 10⁻⁸ F1-G S D C F E S V E A L R A C M Y 72 9.68 × 10⁻⁷ 6.84 ×10⁻⁷ F1-H M D C L A S V E A L K W C M Y 80 1.47 × 10⁻⁷ 7.02 × 10⁻⁸ F2-AG G C A S D L A G F R Y C W E 106 1.33 × 10⁻⁶ 6.64 × 10⁻⁷ F2-B V R C A YD A G G S R Y C W E 97 2.46 × 10⁻⁵ 0.0003873 F2-C V K C A R D L S G F VY C W A 113 0.010019  1.5148   F2-D V R C A W D L G G R A Y C W A 1100.0002493 0.015932  F2-E L G C A S D L A G F W Y C W A 107 5.54 × 10⁻⁷2.09 × 10⁻⁷ F2-F F R C A W E L G W L Y V L L G 120 0.0002087 0.0039645F2-G A S C V A D A G G G G Y C W D 102 9.86 × 10⁻⁷ 5.95 × 10⁻⁷ F2-H W RC V D D L G G F Q Y C W A 112 3.19 × 10⁻⁷ 7.43 × 10⁻⁸ F2-I V T C A A D AL G F L Y C W E 104 1.03 × 10⁻⁶ 4.06 × 10⁻⁷ F2-J L R C A E D L G G Y F YC W A 111 2.12 × 10⁻⁶ 1.33 × 10⁻⁶ F2-K Y R C A T D L A G F S Y C W A 1081.45 × 10⁻⁶ 3.86 × 10⁻⁷ F2-L A R C A R D A G G F Y Y C W A 98 4.36 ×10⁻⁶ 5.28 × 10⁻⁶ F2-M W R C A Q D A G G W T Y C W A 95 1.04 × 10⁻⁶ F2-NV R C V A D A G G F L Y C W A 101 1.18 × 10⁻⁶

TABLE 5 BP1 BP3 Insulin IGF-1 MCF-7 Synthetic IGF-1 binding Comp CompKIRA KIRA binding Peptide Phage (μM) (μM) (μM) (μM) (μM) F1-PDECLMSVEALKNCMGG NT — — 183 400 — (SEQ ID NO: 121) F1-1 RNCFESVAALRRCMYG1.08 × 10⁻⁷M 1.4 2.93 5.48 84.9 5.12 (SEQ ID NO: 2) F1-2MDCLASVEALKWCMYG 1.47 × 10⁻⁷M 15.5 9.20 32.6 20 15.3 (SEQ ID NO: 3) F1-3FECLTSVEALRGCMYG 2.86 × 10⁻⁷M 11.65 12.84 66.1 102 45.7 (SEQ ID NO: 4)F2-P ARCVVDAGGTWYCWAG NT — — 300 NT — (SEQ ID NO: 122) F2-1LGCASDLAGFWYCWAG 5.54 × 10⁻⁷M — — 40.41 NT — (SEQ ID NO: 5) F2-2WRCVDDLGGFQYCWAG 3.19 × 10⁻⁷M — — 93.6 130 — (SEQ ID NO: 6)

Example 2

Structure Determination of IGF-F1-1 by NMR

¹H NMR data were collected on peptide IGF-F1-1 either in pure H₂Osolution (30° C., pH 5.1 and 5.0 millimolar concentration) or in H₂Ocontaining 6% (v/v) d₆-DMSO (40° C., pH 5.2 and at a concentration of6.7 millimolar). In addition to one-dimensional spectra, two-dimensionaldouble-quantum-filtered correlation spectroscopy (2QF-COSY), totalcorrelation spectra (TOCSY), and rotating-frame Overhauser effectspectra (ROESY) were collected. The experiments were recorded asdescribed by Cavanagh et al. in Protein NMR Spectroscopy Principles andPractice (Academic Press, San Diego; ISBN 0-12-164490-1, 1995), exceptthat pulsed-field gradients were used for coherence selection in the2QF-COSY (van Zijl et al., J. Magn. Reson., 113A: 265-270 (1995)) andexcitation sculpting was used to suppress the water resonance in theTOCSY and ROESY experiments (Hwang and Shaka, J. Magn. Reson., 112A:275-279 (1995)). After lyophilization and dissolution of the peptide in²H₂O, a 2D ROESY spectrum (Cavanagh et al.,.supra) and a COSY spectrumwith a 35° mixing pulse (Cavanagh et al, supra) were acquired. Complete1H resonance assignments were obtained from these data by standardmethods (Wütthrich, in NMR of proteins and nucleic acids (John Wiley &Sons, New York; ISBN 0-471-82893-9, 1986) and are listed in Table 6.

Evidence of a well defined three-dimensional structure for IGF-F1-1 wasobtained from the following:

(1) Scalar coupling constants between amide and alpha protons (obtainedfrom the 2QF-COSY spectrum) are distinct from the averaged valuesobserved in unstructured peptides. The values less than 6.0 Hz for Glu5,Ser6, Ala8, Ala9, Leu10, Arg11, Arg12, and Cys13 are indicative of ahelical conformation for these residues. The value of 8.3 Hz observedfor Phe4 is indicative of an extended conformation for this residue.

(2) Scalar coupling constants were also measured between alpha and betaprotons in the COSY-35 spectrum. These data indicate that the sidechains of Cys3, Phe4, Ser6 and Cys13 have fixed chi-1 angles; i.e.,these side chains do not sample the range of chi-1 rotamers that arepopulated in unstructured peptides.

(3) Peaks in the ROESY spectra indicate that there are manyproton-proton contacts (<5 Å) between residues that are not adjacent inthe primary sequence. These can only occur if the peptide folds up intoa well-defined structure. Contacts between residues at position i andi+3 in the primary sequence are prevalent between Val6 and Arg12,consistent with the presence of a helix in this region. Many contactsare observed between the aromatic side chain of Phe4 and the methylgroups of Val7, Leu10, and Met14, indicating the presence of ahydrophobic patch along one face of the helix.

The NMR data were used to derive restraints that could be used todetermine a three-dimensional model of the IGF-F1-1 structure. Dihedralangle restraints were derived from the amide-alpha and alpha-beta scalarcoupling constants via an appropriate Karplus relationship (Karplus, J.Phys. Chem., 30: 11-15 (1959)). Distance restraints were introducedbetween protons that exhibited a through-space interaction in the ROESYspectrum; the size of the upper bound, and corrections to the upperbound because of peak overlap or resonance degeneracy were as describedby Skelton et al., Biochemistry, 33: 13581-92 (1994). These restraintswere used to generate a family of structures using the program DGII(Havel, Prog. Biophys. Mol. Biol., 56: 43-78 (1991)), which weresubsequently refined by restrained molecular dynamics with the programDiscover (MSI, San Diego) using the AMBER all-atom force field (Weineret al., J. Comput. Chem., 7: 230-252 (1986)). 81 inter-proton distancerestraints (45 between non-sequential residues) and 18 dihedral anglerestraints (14φ and 4χ1) were used in the final calculation. Theresulting structures converged to a single global fold (meanroot-mean-squared deviation from the mean structure of 0.46±0.16 Å and1.51±0.14 Å for backbone or all heavy atoms, respectively, of residues3-13). The best 20 structures (least violation of the input restraints)agreed with the input data very well (no distance restraint violationsgreater than 0.13 Å and no dihedral angle violations greater than 2.0°),and had good covalent geometry as judged by the program PROCHECK(Laskowski et al., J. Appl. Cryst., 26: 283-291). Mean coordinates weregenerated for IGF-F1-1 from the ensemble of 20 structure; energyminimization of these coordinates under the influence of theexperimental restraints produced the minimized mean structure depictedin FIG. 1. The atomic coordinates of the minimized mean are listed inTable 7.

According to the Kabsch and Sander secondary structure algorithm withinPROCHECK (supra), IGF-F1-1 contains a type I reverse turn at residuesCys3-Phe4 and an alpha helix from Val7 to Cys13; Ser6 and Met14 areextensions of the main helix. Hydrogen-bond interactions consistent withthese designations are observed in most of the structures within theensemble. Residues Arg1, Asn2, Tyr15, and Gly16 are not well defined bythe NMR restraints and may be more flexible in solution than the otherresidues. There are extensive hydrophobic contacts between theside-chains of Phe4, Val7, Leu10, and Met14. These residues also pack ontop of the disulfide bond (residues Cys3 and Cys13). The non-bondedinteractions along this face of the helix likely help to stabilize thefolded conformation of the peptide.

IGF-F1-1 was selected from a peptide library displayed on phage.Although selection was based only on the ability of the peptide to bindto IGF-1, the sequence identified also folds up into a stable, compactstructure. Highly structured peptides containing a C-terminal helix havebeen observed in a number of other phage-derived peptide selectionexperiments (Dennis et al., Nature, 404: 465-470 (2000); Lowman et al.,Biochemistry, 37: 8870-8878 (1998)). In these examples the conformationobserved for the peptide in solution is similar to that present whenbound to the target protein. Selection of a peptide that has a stablefold in solution that does not change significantly on binding to itstarget provides an energetic benefit to binding since the associationwill not lead to a loss of conformational entropy. In these twoexamples, hydrophobic residues on one face of the peptide helix provideimportant contacts for binding to the target protein. On the basis ofthese prior findings, it is hypothesized, without being limited to anyone theory, that the hydrophobic patch of residues on the front surfacedepicted in FIG. 1 (Phe4, Val7, Leu10, Met14) likely represents thesurface that IGF-F1-1 uses to bind to or interact with IGF-1. Thus, thestructure of IGF-F1-1 contains information about the particulararrangement of atoms that is necessary for binding to IGF-1. TABLE 6Chemical shifts and coupling constant data for IGF-F1-1^(a) ³J_(HN-) ResH^(N) H^(α) H^(β) Other ³J_(hα-Hβ) Hα  1 Arg — 4.02 1.89* γ = 1.63*; δ =3.18*; *, * —  2 Asn — 4.80 2.84, 2.76 δ = 7.57, 6.95 7.6, 6.5 —  3 Cys8.52 4.37 2.92 , 2.68 4.5, 10.0 6.2  4 Phe 7.98 4.65 3.28, 2.92 δ =7.23, ε = 7.33, ζ = 7.28 4.5, 11.0 8.3  5 Glu 7.82 4.18 2.03* γ =2.28* * * 5.6  6 Ser 7.73 4.57 4.09, 4.00 4.5, 3.0 5.9  7 Val 8.43 3.962.14 γ = 1.06, 0.99 8.0 6.2  8 Ala 8.12 4.13 1.40 — 5.4  9 Ala 7.97 4.131.45 — 5.1 10 Leu 7.87 4.19 1.80, 1.75 γ = 1.62, δ = 0.91, 0.85 *, * 5.111 Arg 8.13 4.12 1.92, 1.87 γ = 1.77, 1.64, δ = 3.19* — 4.7 12 Arg 7.964.26 1.90, 1.84 γ = 1.79, 1.64, δ = 3.19* — 6.1 13 Cys 8.22 4.47 3.35 ,3.08 11.0, 3.5 6.0 14 Met 8.17 4.29 1.85* γ = 2.49, 2.37, ε = 2.10 — 6.615 Tyr 8.03 4.65 3.16, 2.94 δ = 7.16, ε = 6.83 5.5, 10.5 7.9 16 Gly 8.073.95, 3.87 — —^(a)Data obtained from H₂O solution containing 6% (v/v) d6-DMSO at 40°C. and pH 5.1. Stereospecifically assigned prochiral groups areindicated by bold typeface; underlined resonances indicate the proRstereochemistry.*indicates degenerate methylene (methyl) resonances.

TABLE 7 Atomic coordinates of the minimized mean structure of IGF-F1-1REMARK 4 1IGF COMPLIES WITH FORMAT V. 2.0, 31-JAN-2001 ATOM 1 N ARG 19.068 4.862 −21.485 1.00 0.00 N1+ ATOM 2 CA ARG 1 8.948 5.526 −20.1721.00 0.00 C ATOM 3 C ARG 1 7.528 6.018 −19.856 1.00 0.00 C ATOM 4 O ARG1 7.310 6.619 −18.805 1.00 0.00 O ATOM 5 CB ARG 1 10.010 6.628 −20.0051.00 0.00 C ATOM 6 CG ARG 1 10.080 7.674 −21.132 1.00 0.00 C ATOM 7 CDARG 1 8.816 8.524 −21.315 1.00 0.00 C ATOM 8 NE ARG 1 8.348 9.114−20.052 1.00 0.00 N1+ ATOM 9 CZ ARG 1 8.892 10.172 −19.427 1.00 0.00 CATOM 10 NH1 ARG 1 9.953 10.809 −19.942 1.00 0.00 N ATOM 11 NH2 ARG 18.364 10.593 −18.271 1.00 0.00 N ATOM 12 1H ARG 1 8.848 5.519 −22.2191.00 0.00 H ATOM 13 2H ARG 1 10.013 4.526 −21.606 1.00 0.00 H ATOM 14 3HARG 1 8.428 4.081 −21.529 1.00 0.00 H ATOM 15 HA ARG 1 9.169 4.765−19.422 1.00 0.00 H ATOM 16 1HB ARG 1 9.871 7.128 −19.046 1.00 0.00 HATOM 17 2HB ARG 1 10.983 6.135 −19.972 1.00 0.00 H ATOM 18 1HG ARG 110.308 7.179 −22.076 1.00 0.00 H ATOM 19 2HG ARG 1 10.914 8.339 −20.9041.00 0.00 H ATOM 20 1HD ARG 1 9.016 9.313 −22.041 1.00 0.00 H ATOM 212HD ARG 1 8.020 7.906 −21.727 1.00 0.00 H ATOM 22 HE ARG 1 7.549 8.667−19.619 1.00 0.00 H ATOM 23 1HH1 ARG 1 10.354 11.603 −19.465 1.00 0.00 HATOM 24 2HH1 ARG 1 10.356 10.496 −20.813 1.00 0.00 H ATOM 25 1HH2 ARG 17.552 10.124 −17.894 1.00 0.00 H ATOM 26 2HH2 ARG 1 8.756 11.385 −17.7831.00 0.00 H ATOM 27 N ASN 2 6.559 5.760 −20.745 1.00 0.00 N ATOM 28 CAASN 2 5.164 6.118 −20.525 1.00 0.00 C ATOM 29 C ASN 2 4.533 5.260−19.427 1.00 0.00 C ATOM 30 O ASN 2 3.607 5.721 −18.766 1.00 0.00 O ATOM31 CB ASN 2 4.368 5.991 −21.831 1.00 0.00 C ATOM 32 CG ASN 2 4.188 4.536−22.261 1.00 0.00 C ATOM 33 OD1 ASN 2 5.063 3.972 −22.923 1.00 0.00 OATOM 34 ND2 ASN 2 3.065 3.920 −21.884 1.00 0.00 N ATOM 35 H ASN 2 6.7795.264 −21.597 1.00 0.00 H ATOM 36 HA ASN 2 5.119 7.164 −20.219 1.00 0.00H ATOM 37 1HB ASN 2 4.884 6.539 −22.621 1.00 0.00 H ATOM 38 2HB ASN 23.386 6.445 −21.691 1.00 0.00 H ATOM 39 1HD2 ASN 2 2.912 2.957 −22.1471.00 0.00 H ATOM 40 2HD2 ASN 2 2.376 4.408 −21.323 1.00 0.00 H ATOM 41 NCYS 3 5.015 4.019 −19.260 1.00 0.00 N ATOM 42 CA CYS 3 4.420 2.986−18.419 1.00 0.00 C ATOM 43 C CYS 3 3.982 3.500 −17.051 1.00 0.00 C ATOM44 O CYS 3 2.868 3.218 −16.624 1.00 0.00 O ATOM 45 CB CYS 3 5.389 1.810−18.264 1.00 0.00 C ATOM 46 SG CYS 3 5.740 0.927 −19.807 1.00 0.00 SATOM 47 H CYS 3 5.786 3.732 −19.845 1.00 0.00 H ATOM 48 HA CYS 3 3.5332.615 −18.932 1.00 0.00 H ATOM 49 1HB CYS 3 6.329 2.151 −17.829 1.000.00 H ATOM 50 2HB CYS 3 4.937 1.097 −17.575 1.00 0.00 H ATOM 51 N PHE 44.839 4.271 −16.376 1.00 0.00 N ATOM 52 CA PHE 4 4.620 4.668 −14.9911.00 0.00 C ATOM 53 C PHE 4 3.808 5.963 −14.883 1.00 0.00 C ATOM 54 OPHE 4 3.418 6.342 −13.781 1.00 0.00 O ATOM 55 CB PHE 4 5.979 4.775−14.291 1.00 0.00 C ATOM 56 CG PHE 4 6.802 3.507 −14.430 1.00 0.00 CATOM 57 CD1 PHE 4 6.472 2.370 −13.668 1.00 0.00 C ATOM 58 CD2 PHE 47.860 3.443 −15.357 1.00 0.00 C ATOM 59 CE1 PHE 4 7.157 1.160 −13.8721.00 0.00 C ATOM 60 CE2 PHE 4 8.549 2.234 −15.557 1.00 0.00 C ATOM 61 CZPHE 4 8.192 1.090 −14.821 1.00 0.00 C ATOM 62 H PHE 4 5.729 4.495−16.796 1.00 0.00 H ATOM 63 HA PHE 4 4.058 3.890 −14.476 1.00 0.00 HATOM 64 1HB PHE 4 6.529 5.618 −14.712 1.00 0.00 H ATOM 65 2HB PHE 45.817 4.977 −13.231 1.00 0.00 H ATOM 66 HD1 PHE 4 5.675 2.417 −12.9411.00 0.00 H ATOM 67 HD2 PHE 4 8.131 4.312 −15.938 1.00 0.00 H ATOM 68HE1 PHE 4 6.888 0.284 −13.301 1.00 0.00 H ATOM 69 HE2 PHE 4 9.351 2.182−16.279 1.00 0.00 H ATOM 70 HZ PHE 4 8.717 0.159 −14.980 1.00 0.00 HATOM 71 N GLU 5 3.517 6.612 −16.018 1.00 0.00 N ATOM 72 CA GLU 5 2.5867.725 −16.118 1.00 0.00 C ATOM 73 C GLU 5 1.192 7.161 −16.410 1.00 0.00C ATOM 74 O GLU 5 0.253 7.395 −15.652 1.00 0.00 O ATOM 75 CB GLU 5 3.0488.689 −17.220 1.00 0.00 C ATOM 76 CG GLU 5 4.445 9.252 −16.928 1.00 0.00C ATOM 77 CD GLU 5 4.950 10.091 −18.096 1.00 0.00 C ATOM 78 OE1 GLU 55.644 9.505 −18.955 1.00 0.00 O ATOM 79 OE2 GLU 5 4.644 11.303 −18.1071.00 0.00 O1− ATOM 80 H GLU 5 3.862 6.243 −16.894 1.00 0.00 H ATOM 81 HAGLU 5 2.556 8.277 −15.177 1.00 0.00 H ATOM 82 1HB GLU 5 3.068 8.176−18.181 1.00 0.00 H ATOM 83 2HB GLU 5 2.340 9.517 −17.287 1.00 0.00 HATOM 84 1HG GLU 5 5.155 8.440 −16.766 1.00 0.00 H ATOM 85 2HG GLU 54.407 9.865 −16.027 1.00 0.00 H ATOM 86 N SER 6 1.070 6.406 −17.510 1.000.00 N ATOM 87 CA SER 6 −0.159 5.760 −17.938 1.00 0.00 C ATOM 88 C SER 6−0.348 4.451 −17.166 1.00 0.00 C ATOM 89 O SER 6 0.164 3.409 −17.5751.00 0.00 O ATOM 90 CB SER 6 −0.111 5.533 −19.456 1.00 0.00 C ATOM 91 OGSER 6 1.085 4.881 −19.830 1.00 0.00 O ATOM 92 H SER 6 1.892 6.238−18.076 1.00 0.00 H ATOM 93 HA SER 6 −1.008 6.416 −17.739 1.00 0.00 HATOM 94 1HB SER 6 −0.966 4.929 −19.763 1.00 0.00 H ATOM 95 2HB SER 6−0.156 6.494 −19.969 1.00 0.00 H ATOM 96 HG SER 6 1.246 4.174 −19.1981.00 0.00 H ATOM 97 N VAL 7 −1.099 4.521 −16.059 1.00 0.00 N ATOM 98 CAVAL 7 −1.390 3.409 −15.156 1.00 0.00 C ATOM 99 C VAL 7 −1.852 2.163−15.922 1.00 0.00 C ATOM 100 O VAL 7 −1.406 1.058 −15.622 1.00 0.00 OATOM 101 CB VAL 7 −2.428 3.844 −14.104 1.00 0.00 C ATOM 102 CG1 VAL 7−1.886 4.995 −13.243 1.00 0.00 C ATOM 103 CG2 VAL 7 −2.804 2.681 −13.1741.00 0.00 C ATOM 104 H VAL 7 −1.468 5.426 −15.805 1.00 0.00 H ATOM 105HA VAL 7 −0.470 3.161 −14.627 1.00 0.00 H ATOM 106 HB VAL 7 −3.334 4.185−14.610 1.00 0.00 H ATOM 107 1HG1 VAL 7 −0.965 4.688 −12.747 1.00 0.00 HATOM 108 2HG1 VAL 7 −1.687 5.877 −13.850 1.00 0.00 H ATOM 109 3HG1 VAL 7−2.622 5.265 −12.485 1.00 0.00 H ATOM 110 1HG2 VAL 7 −3.480 3.035−12.395 1.00 0.00 H ATOM 111 2HG2 VAL 7 −3.311 1.892 −13.729 1.00 0.00 HATOM 112 3HG2 VAL 7 −1.907 2.272 −12.706 1.00 0.00 H ATOM 113 N ALA 8−2.737 2.343 −16.910 1.00 0.00 N ATOM 114 CA ALA 8 −3.245 1.267 −17.7521.00 0.00 C ATOM 115 C ALA 8 −2.110 0.454 −18.383 1.00 0.00 C ATOM 116 OALA 8 −2.138 −0.775 −18.339 1.00 0.00 O ATOM 117 CB ALA 8 −4.159 1.854−18.830 1.00 0.00 C ATOM 118 H ALA 8 −3.065 3.279 −17.099 1.00 0.00 HATOM 119 HA ALA 8 −3.844 0.601 −17.129 1.00 0.00 H ATOM 120 1HB ALA 8−4.562 1.050 −19.447 1.00 0.00 H ATOM 121 2HB ALA 8 −4.986 2.389 −18.3611.00 0.00 H ATOM 122 3HB ALA 8 −3.600 2.544 −19.463 1.00 0.00 H ATOM 123N ALA 9 −1.110 1.137 −18.956 1.00 0.00 N ATOM 124 CA ALA 9 0.041 0.493−19.573 1.00 0.00 C ATOM 125 C ALA 9 0.921 −0.183 −18.521 1.00 0.00 CATOM 126 O ALA 9 1.383 −1.297 −18.755 1.00 0.00 O ATOM 127 CB ALA 90.846 1.510 −20.383 1.00 0.00 C ATOM 128 H ALA 9 −1.117 2.146 −18.9131.00 0.00 H ATOM 129 HA ALA 9 −0.321 −0.267 −20.268 1.00 0.00 H ATOM 1301HB ALA 9 1.641 0.997 −20.925 1.00 0.00 H ATOM 131 2HB ALA 9 0.198 2.019−21.097 1.00 0.00 H ATOM 132 3HB ALA 9 1.294 2.241 −19.714 1.00 0.00 HATOM 133 N LEU 10 1.138 0.487 −17.377 1.00 0.00 N ATOM 134 CA LEU 101.939 0.017 −16.244 1.00 0.00 C ATOM 135 C LEU 10 1.692 −1.468 −15.9591.00 0.00 C ATOM 136 O LEU 10 2.636 −2.251 −15.870 1.00 0.00 O ATOM 137CB LEU 10 1.618 0.868 −15.001 1.00 0.00 C ATOM 138 CG LEU 10 2.813 1.119−14.068 1.00 0.00 C ATOM 139 CD1 LEU 10 2.377 2.089 −12.963 1.00 0.00 CATOM 140 CD2 LEU 10 3.346 −0.165 −13.427 1.00 0.00 C ATOM 141 H LEU 100.724 1.407 −17.290 1.00 0.00 H ATOM 142 HA LEU 10 2.986 0.165 −16.5071.00 0.00 H ATOM 143 1HB LEU 10 1.267 1.843 −15.331 1.00 0.00 H ATOM 1442HB LEU 10 0.812 0.410 −14.427 1.00 −0.00 H ATOM 145 HG LEU 10 3.6231.579 −14.634 1.00 0.00 H ATOM 146 1HD1 LEU 10 3.221 2.317 −12.311 1.000.00 H ATOM 147 2HD1 LEU 10 2.014 3.019 −13.402 1.00 0.00 H ATOM 1483HD1 LEU 10 1.580 1.643 −12.368 1.00 0.00 H ATOM 149 1HD2 LEU 10 2.531−0.720 −12.963 1.00 0.00 H ATOM 150 2HD2 LEU 10 4.088 0.082 −12.668 1.000.00 H ATOM 151 3HD2 LEU 10 3.828 −0.779 −14.184 1.00 0.00 H ATOM 152 NARG 11 0.411 −1.838 −15.843 1.00 0.00 N ATOM 153 CA ARG 11 −0.061 −3.183−15.534 1.00 0.00 C ATOM 154 C ARG 11 0.635 −4.232 −16.405 1.00 0.00 CATOM 155 O ARG 11 1.279 −5.138 −15.881 1.00 0.00 O ATOM 156 CB ARG 11−1.584 −3.244 −15.715 1.00 0.00 C ATOM 157 CG ARG 11 −2.312 −2.303−14.745 1.00 0.00 C ATOM 158 CD ARG 11 −3.799 −2.197 −15.096 1.00 0.00 CATOM 159 NE ARG 11 −4.441 −1.098 −14.363 1.00 0.00 N1+ ATOM 160 CZ ARG11 −4.800 −1.128 −13.069 1.00 0.00 C ATOM 161 NH1 ARG 11 −4.608 −2.225−12.323 1.00 0.00 N ATOM 162 NH2 ARG 11 −5.357 −0.043 −12.515 1.00 0.00N ATOM 163 H ARG 11 −0.290 −1.121 −15.970 1.00 0.00 H ATOM 164 HA ARG 110.170 −3.395 −14.488 1.00 0.00 H ATOM 165 1HB ARG 11 −1.828 −2.967−16.742 1.00 0.00 H ATOM 166 2HB ARG 11 −1.929 −4.264 −15.539 1.00 0.00H ATOM 167 1HG ARG 11 −1.890 −1.303 −14.801 1.00 0.00 H ATOM 168 2HG ARG11 −2.191 −2.670 −13.726 1.00 0.00 H ATOM 169 1HD ARG 11 −4.303 −3.144−14.896 1.00 0.00 H ATOM 170 2HD ARG 11 −3.896 −1.977 −16.160 1.00 0.00H ATOM 171 HE ARG 11 −4.603 −0.248 −14.884 1.00 0.00 H ATOM 172 2HH1 ARG11 −4.184 −3.045 −12.731 1.00 0.00 H ATOM 173 1HH1 ARG 11 −4.882 −2.234−11.351 1.00 0.00 H ATOM 174 1HH2 ARG 11 −5.631 −0.053 −11.543 1.00 0.00H ATOM 175 2HH2 ARG 11 −5.505 0.789 −13.068 1.00 0.00 H ATOM 176 N ARG12 0.521 −4.095 −17.732 1.00 0.00 N ATOM 177 CA ARG 12 1.150 −5.005−18.679 1.00 0.00 C ATOM 178 C ARG 12 2.668 −4.836 −18.713 1.00 0.00 CATOM 179 O ARG 12 3.387 −5.828 −18.801 1.00 0.00 O ATOM 180 CB ARG 120.576 −4.808 −20.086 1.00 0.00 C ATOM 181 CG ARG 12 −0.805 −5.459−20.231 1.00 0.00 C ATOM 182 CD ARG 12 −1.215 −5.535 −21.705 1.00 0.00 CATOM 183 NE ARG 12 −0.296 −6.395 −22.465 1.00 0.00 N1+ ATOM 184 C ARG 12−0.331 −6.587 −23.792 1.00 0.00 C ATOM 185 NH1 ARG 12 −1.292 −6.032−24.544 1.00 0.00 N ATOM 186 NH2 ARG 12 0.613 −7.344 −24.367 1.00 0.00 NATOM 187 H ARG 12 0.007 −3.307 −18.101 1.00 0.00 H ATOM 188 HA ARG 120.942 −6.025 −18.370 1.00 0.00 H ATOM 189 1HB ARG 12 0.523 −3.747−20.333 1.00 0.00 H ATOM 190 2HB ARG 12 1.262 −5.284 −20.783 1.00 0.00 HATOM 191 1HG ARG 12 −0.778 −6.473 −19.832 1.00 0.00 H ATOM 192 2HG ARG12 −1.541 −4.879 −19.673 1.00 0.00 H ATOM 193 1HD ARG 12 −2.223 −5.949−21.769 1.00 0.00 H ATOM 194 2HD ARG 12 −1.216 −4.530 −22.129 1.00 0.00H ATOM 195 HE ARG 12 0.445 −6.844 −21.942 1.00 0.00 H ATOM 196 2HH1 ARG12 −2.004 −5.462 −24.111 1.00 0.00 H ATOM 197 1HH1 ARG 12 −1.307 −6.182−25.542 1.00 0.00 H ATOM 198 1HH2 ARG 12 1.348 −7.745 −23.800 1.00 0.00H ATOM 199 2HH2 ARG 12 0.603 −7.503 −25.364 1.00 0.00 H ATOM 200 N CYS13 3.142 −3.586 −18.683 1.00 0.00 N ATOM 201 CA CYS 13 4.531 −3.206−18.914 1.00 0.00 C ATOM 202 C CYS 13 5.525 −3.948 −18.013 1.00 0.00 CATOM 203 O CYS 13 6.661 −4.171 −18.428 1.00 0.00 O ATOM 204 CB CYS 134.649 −1.684 −18.783 1.00 0.00 C ATOM 205 SG CYS 13 6.253 −0.953 −19.2021.00 0.00 S ATOM 206 H CYS 13 2.476 −2.833 −18.584 1.00 0.00 H ATOM 207HA CYS 13 4.770 −3.463 −19.947 1.00 0.00 H ATOM 208 1HB CYS 13 3.918−1.242 −19.461 1.00 0.00 H ATOM 209 2HB CYS 13 4.396 −1.389 −17.766 1.000.00 H ATOM 210 N MET 14 5.105 −4.380 −16.816 1.00 0.00 N ATOM 211 CAMET 14 5.942 −5.147 −15.894 1.00 0.00 C ATOM 212 C MET 14 6.053 −6.635−16.274 1.00 0.00 C ATOM 213 O MET 14 6.252 −7.476 −15.399 1.00 0.00 OATOM 214 CB MET 14 5.406 −4.976 −14.464 1.00 0.00 C ATOM 215 CG MET 145.447 −3.513 −14.009 1.00 0.00 C ATOM 216 SD MET 14 4.877 −3.219 −12.3131.00 0.00 S ATOM 217 CE MET 14 3.147 −3.748 −12.439 1.00 0.00 C ATOM 218H MET 14 4.157 −4.176 −16.530 1.00 0.00 H ATOM 219 HA MET 14 6.954−4.744 −15.928 1.00 0.00 H ATOM 220 1HB MET 14 4.382 −5.349 −14.430 1.000.00 H ATOM 221 2HB MET 14 6.016 −5.556 −13.770 1.00 0.00 H ATOM 222 1HGMET 14 4.841 −2.904 −14.676 1.00 0.00 H ATOM 223 2HG MET 14 6.478 −3.163−14.074 1.00 0.00 H ATOM 224 1HE MET 14 3.096 −4.826 −12.584 1.00 0.00 HATOM 225 2HE MET 14 2.627 −3.490 −11.517 1.00 0.00 H ATOM 226 3HE MET 142.665 −3.245 −13.277 1.00 0.00 H ATOM 227 N TYR 15 5.966 −6.955 −17.5711.00 0.00 N ATOM 228 CA TYR 15 6.208 −8.276 −18.144 1.00 0.00 C ATOM 229C TYR 15 6.195 −8.190 −19.673 1.00 0.00 C ATOM 230 O TYR 15 7.091 −8.715−20.332 1.00 0.00 O ATOM 231 CB TYR 15 5.219 −9.338 −17.628 1.00 0.00 CATOM 232 CG TYR 15 3.737 −9.035 −17.779 1.00 0.00 C ATOM 233 CD1 TYR 153.054 −9.396 −18.956 1.00 0.00 C ATOM 234 CD2 TYR 15 3.028 −8.436−16.720 1.00 0.00 C ATOM 235 CE1 TYR 15 1.667 −9.198 −19.058 1.00 0.00 CATOM 236 CE2 TYR 15 1.635 −8.274 −16.808 1.00 0.00 C ATOM 237 CZ TYR 150.952 −8.664 −17.972 1.00 0.00 C ATOM 238 OH TYR 15 −0.402 −8.517−18.047 1.00 0.00 O ATOM 239 H TYR 15 5.831 −6.199 −18.224 1.00 0.00 HATOM 240 HA TYR 15 7.209 −8.590 −17.845 1.00 0.00 H ATOM 241 1HB TYR 155.430 −10.269 −18.157 1.00 0.00 H ATOM 242 2HB TYR 15 5.422 −9.532−16.576 1.00 0.00 H ATOM 243 HD1 TYR 15 3.587 −9.844 −19.782 1.00 0.00 HATOM 244 HD2 TYR 15 3.541 −8.135 −15.819 1.00 0.00 H ATOM 245 HE1 TYR 151.151 −9.477 −19.966 1.00 0.00 H ATOM 246 HE2 TYR 15 1.089 −7.854−15.976 1.00 0.00 H ATOM 247 HH TYR 15 −0.771 −8.820 −18.879 1.00 0.00 HATOM 248 N GLY 16 5.175 −7.530 −20.233 1.00 0.00 N ATOM 249 CA GLY 164.972 −7.394 −21.664 1.00 0.00 C ATOM 250 C GLY 16 3.630 −6.713 −21.9201.00 0.00 C ATOM 251 O GLY 16 2.619 −7.446 −21.996 1.00 0.00 O ATOM 252OXT GLY 16 3.636 −5.467 −22.024 1.00 0.00 O1− ATOM 253 H GLY 16 4.476−7.124 −19.626 1.00 0.00 H ATOM 254 1HA GLY 16 5.777 −6.795 −22.091 1.000.00 H ATOM 255 2HA GLY 16 4.974 −8.380 −22.131 1.00 0.00 H TER

The present invention has of necessity been discussed herein byreference to certain specific methods and materials. It is to beunderstood that the discussion of these specific methods and materialsin no way constitutes any limitation on the scope of the presentinvention, which extends to any and all alternative materials andmethods suitable for accomplishing the objectives of the presentinvention.

1. A peptide comprising the sequence:(Xaa)₁(Xaa)₂Cys(Xaa)₃(Xaa)₄Asp(Xaa)₅(Xaa)₆Gly(Xaa)₇(Xaa)₈TyrCysTrp(Xaa)₉(SEQ ID NO:5), where (Xaa)₁, (Xaa)₄, and (Xaa)₈ are an amino acid,(Xaa)₂ is Arg, Lys, Gly, Ser, or Thr, (Xaa)₃ is Ala or Val, (Xaa)₅ isAla or Leu, (Xaa)₆ is Ala, Gly, or Leu, (Xaa)₇ is Phe, Tyr, Trp, or Gly,and (Xaa)₉ is Glu, Asp, Ala, or Gly.
 2. The peptide of claim 1 whereinthe amino acids in the peptide are all L-amino acids.
 3. The peptide ofclaim 1 further comprising a glycine residue after (Xaa)₉.
 4. Thepeptide of claim 1 wherein (Xaa)₂ is Gly, Ser, Arg, or Thr, and (Xaa)₉is Glu, Ala, or Asp.
 5. The peptide of claim 1 wherein (Xaa)₂ is Glu orArg, (Xaa)₅ is Leu, (Xaa)₆ is Ala or Gly, (Xaa)₇ is Phe, and (Xaa)₉ isAla.
 6. The peptide of claim 1 that comprises the sequenceLGCASDLAGFWYCWAG (SEQ ID NO:6) or WRCVDDLGGFQYCWAG (SEQ ID NO:7).
 7. Thepeptide of claim 1 that is conjugated to a cytotoxic agent orpolyethylene glycol.